Methods of treatment and prevention using haloaryl substituted aminopurines

ABSTRACT

Provided herein are Aminopurine Compounds having the following structure:  
                 
 
     wherein R 1 , R 2  and R 3  are as defined herein, compositions comprising an effective amount of an Aminopurine Compound and methods for treating or preventing cancer, a cardiovascular disease, a renal disease, an autoimmune condition, an inflammatory condition, macular degeneration, ischemia-reperfusion injury, pain and related syndromes, disease-related wasting, an asbestos-related condition, pulmonary hypertension or a condition treatable or preventable by inhibition of the JNK pathway comprising administering an effective amount of an Aminopurine Compound to a patient in need thereof.

This application is a continuation-in-part of U.S. application Ser. No.11/411,413, filed Apr. 26, 2006, which is a continuation-in-part of U.S.application Ser. No. 11/332,617, filed Jan. 12, 2006, which claims thebenefit of U.S. provisional application No. 60/643,796, filed Jan. 13,2005, and U.S. provisional application No. 60/709,980, filed Aug. 19,2005, the contents of each is incorporated by reference herein in theirentirety.

1. FIELD

Provided herein are certain amino-substituted purine compounds,compositions comprising an effective amount of such compounds andmethods for treating or preventing cancer, a cardiovascular disease, arenal disease, an autoimmune condition, an inflammatory condition,macular degeneration, ischemia-reperfusion injury, pain and relatedsyndromes, disease-related wasting, an asbestos-related condition,pulmonary hypertension, central nervous system (CNS) injury/damage or acondition treatable or preventable by inhibition of a kinase pathwaycomprising administering an effective amount of such aminopurinecompounds to a patient in need thereof.

2. BACKGROUND

The connection between abnormal protein phosphorylation and the cause orconsequence of diseases has been known for over 20 years. Accordingly,protein kinases have become a very important group of drug targets. SeeCohen, Nature, 1:309-315 (2002). Various protein kinase inhibitors havebeen used clinically in the treatment of a wide variety of diseases,such as cancer and chronic inflammatory diseases, including diabetes andstroke. See Cohen, Eur. J. Biochem., 268:5001-5010 (2001).

The protein kinases are a large and diverse family of enzymes thatcatalyze protein phosphorylation and play a critical role in cellularsignaling. Protein kinases may exert positive or negative regulatoryeffects, depending upon their target protein. Protein kinases areinvolved in specific signaling pathways which regulate cell functionssuch as, but not limited to, metabolism, cell cycle progression, celladhesion, vascular function, apoptosis, and angiogenesis. Malfunctionsof cellular signaling have been associated with many diseases, the mostcharacterized of which include cancer and diabetes. The regulation ofsignal transduction by cytokines and the association of signal moleculeswith protooncogenes and tumor suppressor genes have been welldocumented. Similarly, the connection between diabetes and relatedconditions, and deregulated levels of protein kinases, has beendemonstrated. See e.g., Sridhar et al. Pharmaceutical Research,17(11):1345-1353 (2000). Viral infections and the conditions relatedthereto have also been associated with the regulation of proteinkinases. Park et al. Cell 101 (7), 777-787 (2000).

Protein kinases can be divided into broad groups based upon the identityof the amino acid(s) that they target (serine/threonine, tyrosine,lysine, and histidine). For example, tyrosine kinases include receptortyrosine kinases (RTKs), such as growth factors and non-receptortyrosine kinases, such as the src kinase family. There are alsodual-specific protein kinases that target both tyrosine andserine/threonine, such as cyclin dependent kinases (CDKs) andmitogen-activated protein kinases (MAPKs). Any particular cell containsmany protein kinases, some of which phosphorylate other protein kinases.Some protein kinases phosphorylate many different proteins, othersphosphorylate only a single protein. Not surprisingly, there arenumerous classes of protein kinases. Upon receiving a signal, someproteins may also undergo auto-phosphorylation.

The protein tyrosine kinases (PTKs) compose a large family of kinasesthat regulate cell to cell signals involved in growth, differentiation,adhesion, motility, and death. Robinson et al., Oncogene 19:5548-5557(2000). Members of the tyrosine kinase include, but are not limited to,Yes, BMX, Syk, EphA1, FGFR3, RYK, MUSK, JAK1 and EGFR. Tyrosine kinasesare distinguished into two classes, i.e., the receptor type andnon-receptor type tyrosine kinases. Interestingly, the entire of familyof tyrosine kinases is quite large—consisting of at least 90characterized kinases with at least 58 receptor type and at least 32nonreceptor type kinases comprising at least 30 total subfamilies.Robinson et al., Oncogene 19:5548-5557 (2000). Tyrosine kinases havebeen implicated in a number of diseases in humans, including diabetesand cancer. Robinson et al. at page 5548. Tyrosine kinases are ofteninvolved in most forms of human malignancies and have been linked to awide variety of congenital syndromes. Robertson et al., Trends Genet.16:265-271 (2000).

The non-receptor tyrosine kinases represent a group of intracellularenzymes that lack extracellular and transmembrane sequences. Currently,over 32 families of non-receptor tyrosine kinases have been identified.Robinson et al., Oncogene 19:5548-5557 (2000). Examples are Src, Btk,Csk, ZAP70, Kak families. In particular, the Src family of non-receptortyrosine kinase family is the largest, consisting of Src, Yes, Fyn, Lyn,Lck, Blk, Hck, Fgr and Yrk protein tyrosine kinases. The Src family ofkinases have been linked to oncogenesis, cell proliferation and tumorprogression. A detailed discussion of non-receptor protein tyrosinekinases is available in Oncogene 8:2025-2031 (1993). Many of theseprotein tyrosine kinases have been found to be involved in cellularsignaling pathways involved in various pathological conditions includingbut not limited to cancer and hyperproliferative disorders and immunedisorders.

The cyclin dependent kinases CDKs represent a group of intracellularenzymes that control progression through the cell cycle and haveessential roles in cell proliferation. See Cohen, Nature, 1:309-315(2002). Examples of CDKs include, but are not limited to, cyclindependent kinase 2 (CDK2), cyclin dependent kinase 7 (CDK7), cyclindependent kinase 6 (CDK6) and cell division control 2 protein (CDC2).CDKs have been implicated in the regulation of transitions betweendifferent phases of the cell cycle, such as the progression from aquiescent stage in G₁ (the gap between mitosis and the onset of DNAreplication for a new round of cell division) to S (the period of activeDNA synthesis), or the progression from G₂ to M phase, in which activemitosis and cell division occur. See e.g., the articles compiled inScience, vol. 274 (1996), pp. 1643-1677; and Ann. Rev. Cell Dev Biol.,vol. 13 (1997), pp. 261-291. CDK complexes are formed throughassociation of a regulatory cyclin subunit (e.g., cyclin A, B1, B2, D1,D2, D3, and E) and a catalytic kinase subunit (e.g., cdc2 (CDK1), CDK2,CDK4, CDK5, and CDK6). As the name implies, CDKs display an absolutedependence on the cyclin subunit in order to phosphorylate their targetsubstrates, and different kinase/cyclin pairs function to regulateprogression through specific portions of the cell cycle. CDKs have beenimplicated in various disease states, including but not limited to,those displaying the cancer phenotype, various neoplastic disorders andin neurological disorders. Hunter, Cell 100:113-127 (2000).

The mitogen activated protein (MAP) kinases participate in thetransduction of signals to the nucleus of the cell in response toextracellular stimuli. Examples of MAP kinases include, but are notlimited to, mitogen activated protein kinase 3 (MAPK3),mitogen-activated protein kinase 1 (ERK2), mitogen-activated proteinkinase 7 (MAPK7), mitogen-activated protein kinase 8 (JNK1),mitogen-activated protein kinase 14 (p38 alpha), mitogen-activatedprotein kinase 10 (MAPK10), JNK3 alpha protein kinase, stress-activatedprotein kinase JNK2 and mitogen-activated protein kinase 14 (MAPK14).MAP kinases are a family of proline-directed serine/threonine kinasesthat mediate signal transduction from extracellular receptors or heathshock, or UV radiation. See Sridhar et al., Pharmaceutical Research,17:11 1345-1353 (2000). MAP kinases activate through the phosphorylationof theonine and tyrosine by dual-specificity protein kinases, includingtyrosine kinases such as growth factors. Cell proliferation anddifferentiation have been shown to be under the regulatory control ofmultiple MAP kinase cascades. See Sridhar et al., PharmaceuticalResearch, 17:11 1345-1353 (2000). As such, the MAP kinase pathway playscritical roles in a number of disease states. For example, defects inactivities of MAP kinases have been shown to lead to aberrant cellproliferation and carcinogenesis. See Hu et al., Cell Growth Differ.11:191-200 (2000); and Das et al., Breast Cancer Res. Treat. 40:141(1996). Moreover, MAP kinase activity has also been implicated ininsulin resistance associated with type-2 diabetes. See Virkamaki etal., J. Clin. Invest. 103:931-943 (1999).

The p90 ribosomal S6 kinases (Rsk) are serine/threonine kinases. The Rskfamily members function in mitogen-activated cell growth andproliferation, differentiation, and cell survival. Examples of membersof the Rsk family of kinases include, but are not limited to, ribosomalprotein S6 kinase, 90 kDa, polypeptide 2 (Rsk3), ribosomal protein S6kinase, 90 kDa, polypeptide 6 (Rsk4), ribosomal protein S6 kinase, 90kDa, polypeptide 3 (Rsk2) and ribosomal protein S6 kinase, 90 kDa,polypeptide 1 (Rsk1/p90Rsk). The Rsk family members are activated byextracellular signal-related kinases 1/2 and phosphoinositide-dependentprotein kinase 1. Frodin and Gammeltoft, Mol. Cell. Endocrinol.151:65-77 (1999). Under basal conditions, RSK kinases are localized inthe cytoplasm of cells and upon stimulation by mitogens, the activated(phosphorylated by extracellular-related kinase) RSK transientlytranslocates to the plasma membrane where they become fully activated.The fully activated RSK phosphorylates substrates that are involved incell growth, proliferation, differentiation, and cell survival. Richardset al., Curr. Biol. 9:810-820 (1999); Richards et al., Mol. Cell. Biol.21:7470-7480 (2001). RSK signaling pathways have also been associatedwith the modulation of the cell cycle. Gross et al., J. Biol. Chem.276(49): 46099-46103 (2001). Current data suggests that small moleculesthat inhibit Rsk may be useful therapeutic agents for the prevention andtreatment of cancer and inflammatory diseases.

Members of the checkpoint protein kinase family are serine/threoninekinases that play an important role in cell cycle progression. Examplesof members of the checkpoint family include, but are not limited to,CHK1 and CHK2. Checkpoints are control systems that coordinate cellcycle progression by influencing the formation, activation andsubsequent inactivation of the cyclin-dependent kinases. Checkpointsprevent cell cycle progression at inappropriate times, maintain themetabolic balance of cells while the cell is arrested, and in someinstances can induce apoptosis (programmed cell death) when therequirements of the checkpoint have not been met. See e.g., O'Connor,Cancer Surveys, 29: 151-182 (1997); Nurse, Cell, 91: 865-867 (1997);Hartwell et al., Science, 266: 1821-1828 (1994); Hartwell et al.,Science, 246: 629-634 (1989). Members of the checkpoint family ofkinases have been implicated in cell proliferative disorders, cancerphenotypes and other diseases related to DNA damage and repair. Kohn,Mol. Biol. Cell 10:2703-2734 (1999); Ohi and Gould, Curr. Opin. CellBiol. 11:267-273 (1999); Peng, et al., Science 277:1501-1505 (1997).

Aurora kinases are a family of multigene mitotic serine-threoninekinases that functions as a class of novel oncogenes. These kinasescomprise aurora-A and aurora-B members. Aurora kinases arehyperactivated and/or over-expressed in several solid tumors includingbut not limited to, breast, ovary, prostate, pancreas, and colorectalcancers. In particular aurora-A is a centrosome kinase that plays animportant role cell cycle progression and cell proliferation. Aurora-Ais located in the 20q13 chromosome region that is frequently amplifiedin several different types of malignant tumors such as colorectal,breast and bladder cancers. There is also a high correlation betweenaurora-A and high histo-prognostic grade aneuploidy, making the kinase apotential prognostic vehicle. Inhibition of aurora kinase activity couldhelp to reduce cell proliferation, tumor growth and potentiallytumorigenesis. A detailed description of aurora kinase function isreviewed in Oncogene 21:6175-6183 (2002).

The Rho-associated coiled-coil-containing protein serine/threoninekinases ROCK-I and ROCK-II are thought to play a major role incytoskeletal dynamics by serving as downstream effectors of the Rho/Racfamily of cytokine- and growth factor-activated small GTPases. ROCKsphosphorylate various substrates, including, but not limited to, myosinlight chain phosphatase, myosin light chain, ezrin-radixin-moesinproteins and LIM (for Lin11, Isl1 and Mec3) kinases. ROCKs also mediatethe formation of actin stress fibers and focal adhesions in various celltypes. ROCKs have an important role in cell migration by enhancing cellcontractility. They are required for tail retraction of monocytes andcancer cells, and a ROCK inhibitor has been used to reduce tumor-celldissemination in vivo. Recent experiments have defined new functions ofROCKs in cells, including centrosome positioning and cell-sizeregulation, which might contribute to various physiological andpathological states. See Nature Reviews Mol. Cell. Biol. 4, 446-456(2003). The ROCK family members are attractive intervention targets fora variety of pathologies, including cancer and cardiovascular disease.For example, Rho kinase inhibitors can be useful therapeutic agents forhypertension, angina pectoris, and asthma. Furthermore, Rho is expectedto play a role in peripheral circulation disorders, arteriosclerosis,inflammation, and autoimmune disease and as such, is a useful target fortherapy.

The 70 kDa ribosomal S6 kinase (p70S6K) is activated by numerousmitogens, growth factors and hormones. Activation of p70S6K occursthrough phosphorylation at a number of sites and the primary target ofthe activated kinase is the 40S ribosomal protein S6, a major componentof the machinery involved in protein synthesis in mammalian cells. Inaddition to its involvement in regulating translation, p70S6K activationhas been implicated in cell cycle control, neuronal celldifferentiation, regulation of cell motility and a cellular responsethat is important in tumor metastases, immunity and tissue repair.Modulation of p70S6 kinase activity may have therapeutic implications indisorders such as cancer, inflammation, and various neuropathies. Adetailed discussion of p70S6K kinases can be found in Prog. Cell CycleRes. 1:21-32 (1995), and Immunol Cell Biol. 78(4):447-51 (2000).

Glycogen synthase kinase 3 (GSK-3) is a ubiquitously expressedconstitutively active serine/threonine kinase that phosphorylatescellular substrates and thereby regulates a wide variety of cellularfunctions, including development, metabolism, gene transcription,protein translation, cytoskeletal organization, cell cycle regulation,and apoptosis. GSK-3 was initially described as a key enzyme involved inglycogen metabolism, but is now known to regulate a diverse array ofcell functions. Two forms of the enzyme, GSK-3α and GSK-3β, have beenpreviously identified. The activity of GSK-3β is negatively regulated byprotein kinase B/Akt and by the Wnt signaling pathway. Small moleculesinhibitors of GSK-3 may, therefore, have several therapeutic uses,including the treatment of neurodegenerative diseases, diabetes type II,bipolar disorders, stroke, cancer, and chronic inflammatory disease.Reviewed in Role of glycogen synthase kinase-3 in cancer: regulation byWnts and other signaling pathways (Adv Cancer Res.; 84:203-29, 2002);Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs fordiabetes, neurodegeneration, cancer, and inflammation (Med Res Rev.;22(4):373-84, 2002); Role of glycogen synthase kinase-3 in thephosphatidylinositol 3-Kinase/Akt cell survival pathway. (J. Biol.Chem., 273(32):19929-32, 1998).

Because protein kinases regulate nearly every cellular process,including metabolism, cell proliferation, cell differentiation, and cellsurvival, they are attractive targets for therapeutic intervention forvarious disease states. For example, cell-cycle control andangiogenesis, in which protein kinases play a pivotal role are cellularprocesses associated with numerous disease conditions such as but notlimited to cancer, inflammatory diseases, abnormal angiogenesis anddiseases related thereto, atherosclerosis, macular degeneration,diabetes, obesity, and pain.

Protein kinases have become attractive targets for the treatment ofcancers. Fabbro et al., Pharmacology & Therapeutics 93:79-98 (2002). Ithas been proposed that the involvement of protein kinases in thedevelopment of human malignancies may occur, by: (1) genomicrearrangements (e.g., BCR-ABL in chronic myelogenous leukemia), (2)mutations leading to constitutively active kinase activity, such asacute myelogenous leukemia and gastrointestinal tumors, (3) deregulationof kinase activity by activation of oncogenes or loss of tumorsuppressor functions, such as in cancers with oncogenic RAS, (4)deregulation of kinase activity by over-expression, as in the case ofEGFR and (5) ectopic expression of growth factors that can contribute tothe development and maintenance of the neoplastic phenotype. Fabbro etal., Pharmacology & Therapeutics 93:79-98 (2002).

Certain cancers are associated with angiogenesis. Angiogenesis is thegrowth of new capillary blood vessels from pre-existing vasculature.Risau, W., Nature 386:671-674 (1997). It has been shown that proteinkinases can contribute to the development and maintenance of theneoplastic phenotype. Fabbro et al., Pharmacology & Therapeutics93:79-98 (2002). For example, VEGF A-D and their four receptors havebeen implicated in phenotypes that involve neovascularization andenhanced vascular permeability, such as tumor angiogenesis andlymphangiogenesis. Matter, A., Drug Discov. Today 6:1005-1023 (2001).

Cardiovascular disease (“CVD”) accounts for nearly one quarter of totalannual deaths worldwide. Vascular disorders such as atherosclerosis andrestenosis result from dysregulated growth of the vessel walls and therestriction of blood flow to vital organs. Various kinase pathways, e.g.JNK, are activated by atherogenic stimuli and regulated through localcytokine and growth factor production in vascular cells. Yang et al.,Immunity 9:575 (1998). Ischemia and ischemia coupled with reperfusion inthe heart, kidney or brain result in cell death and scar formation,which can ultimately lead to congestive heart failure, renal failure orcerebral dysfunction. In organ transplantation, reperfusion ofpreviously ischemic donor organs results in acute leukocyte-mediatedtissue injury and delay of graft function. Ischemia and reperfusionpathways are mediated by various kinases. For example, the JNK pathwayhas been linked to leukocyte-mediated tissue damage. Li et al., Mol.Cell. Biol. 16:5947-5954 (1996). Finally, enhanced apoptosis in cardiactissues has also been linked to kinase activity. Pombo et al., J. Biol.Chem. 269:26546-26551 (1994).

The elucidation of the intricacy of protein kinase pathways and thecomplexity of the relationship and interaction among and between thevarious protein kinases and kinase pathways highlights the importance ofdeveloping pharmaceutical agents capable of acting as protein kinasemodulators, regulators or inhibitors that have beneficial activity onmultiple kinases or multiple kinase pathways.

It has therefore been suggested that due to the complexity ofintracellular signaling cascades of protein kinase pathways, agents thataffect multiple pathways simultaneously may be required for meaningfulclinical activity. Indeed, it is known that some kinase drugs, such asGleevec®, do target several kinases at once. Gleevec® primarily targetsa mutant fusion protein containing the abl kinase, which is created by a9:22 chromosomal translocation event; Gleevec® also targets c-kit, atyrosine kinase implicated in gastrointestinal stromal tumors (GIST).However, in recent clinical trials, patients have developed resistanceto Gleevec® or have shown incomplete response to treatment.

Accordingly, there remains a need for new kinase modulators.

Citation or identification of any reference in Section 2 of thisapplication is not to be construed as an admission that the reference isprior art to the present application.

3. SUMMARY

Provided herein are compounds having the following formula (I):

and pharmaceutically acceptable salts, polymorphs, clathrates, solvates,hydrates, stereoisomers and prodrugs thereof, wherein R¹, R² and R³ areas defined herein.

A compound of formula (I) or a pharmaceutically acceptable salt,clathrate, solvate, hydrate, stereoisomer or prodrug thereof (each beingreferred to herein as an “Aminopurine Compound”) is useful for treatingor preventing cancer, a cardiovascular disease, a renal disease, anautoimmune condition, an inflammatory condition, macular degeneration,ischemia-reperfusion injury, pain and related syndromes, disease-relatedwasting, an asbestos-related condition, pulmonary hypertension, centralnervous system (CNS) injury/damage or a condition treatable orpreventable by inhibition of a kinase pathway, in one embodiment, theJNK pathway.

Further provided herein are compositions comprising an effective amountof an Aminopurine Compound and compositions comprising an effectiveamount of an Aminopurine Compound and a pharmaceutically acceptablecarrier or vehicle. The compositions are useful for treating orpreventing cancer, a cardiovascular disease, a renal disease, anautoimmune condition, an inflammatory condition, macular degeneration,ischemia-reperfusion injury, pain and related syndromes, disease-relatedwasting, an asbestos-related condition, pulmonary hypertension, centralnervous system (CNS) injury/damage or a condition treatable orpreventable by inhibition of a kinase pathway, in one embodiment, theJNK pathway.

Further provided herein are methods for treating or preventing cancer, acardiovascular disease, a renal disease, an inflammatory condition, ametabolic condition, an autoimmune condition, macular degeneration,ischemia-reperfusion injury, pain and related syndromes, disease-relatedwasting, an asbestos-related condition, pulmonary hypertension, centralnervous system (CNS) injury/damage or a condition treatable orpreventable by inhibition of a kinase pathway, in one embodiment, theJNK pathway, comprising administering an effective amount of anAminopurine Compound to a patient in need of the treating or preventing.

In one embodiment, the Aminopurine Compound targets two or more of thefollowing: kinases from the src kinase family, kinases from the Rskkinase family, kinases from the CDK family, kinases from the MAPK kinasefamily, and tyrosine kinases such as Fes, Lyn, and Syk kinases. Theagent may target two or more kinases of the same family, or may targetkinases representing two or more kinase families or classes.

Further provided herein are stents (e.g., stent graft) containing orcoated with an amount of an Aminopurine Compound effective for treatingor preventing a cardiovascular disease or renal disease.

The present embodiments can be understood more fully by reference to thedetailed description and examples, which are intended to exemplifynon-limiting embodiments.

4. DETAILED DESCRIPTION 4.1 Definitions

A “C₁₋₆alkyl” group is a saturated straight chain or branched non-cyclichydrocarbon having from 1 to 6 carbon atoms. Representative—(C₁₋₆alkyls) include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyland -n-hexyl; while saturated branched alkyls include -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylpentyl,3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. A—(C₁₋₆alkyl) group can be substituted or unsubstituted.

An “alkoxy” group is an —O—(C₁₋₆alkyl) group, wherein C₁₋₆alkyl isdefined above, including —OCH₃, —OCH₂CH₃, —O(CH₂)₂CH₃, —O(CH₂)₃CH₃,—O(CH₂)₄CH₃, —O(CH₂)₅CH₃, and the like.

An “alkoxyalkyl” group is a —(C₁₋₆alkylene)-O—(C₁₋₆alkyl) group, whereineach C₁₋₆alkyl is independently a C₁₋₆alkyl group defined above,including —CH₂OCH₃, —CH₂OCH₂CH₃, —(CH₂)₂OCH₂CH₃, —(CH₂)₂O(CH₂)₂CH₃, andthe like.

An “alkylamino” group is a mono-alkylamino or di-alkylamino group, suchas —NH(C₁₋₆alkyl), —N(C₁₋₆alkyl)(C₁₋₆alkyl), —NH(C₃₋₁₀cycloalkyl),—N(C₃₋₁₀cycloalkyl)(C₃₋₁₀cycloalkyl) or —N(C₁₋₆alkyl)(C₃₋₁₀cycloalkyl)wherein each C₁₋₆alkyl and C₃₋₁₀cycloalkyl is independently as definedherein, including, but not limited to, —NHCH₃, —NHCH₂CH₃, —NH(CH₂)₂CH₃,—NH(CH₂)₃CH₃, —NH(CH₂)₄CH₃, —NH(CH₂)₅CH₃, —N(CH₃)₂, —N(CH₂CH₃)₂,—N((CH₂)₂CH₃)₂, and —N(CH₃)(CH₂CH₃).

An “aminocarbonyl” group is a —C(O)NR₂ group, wherein each R isindependently hydrogen or a C₁₋₆alkyl group defined above, wherein eachC₁₋₆alkyl group can be optionally substituted.

An “aminoalkyl” group is a —C(O)NR₂ group, wherein each R isindependently hydrogen or a C₁₋₆alkyl group defined above, wherein eachC₁₋₆alkyl group can be optionally substituted.

An “acylamino” group is a C₁₋₆alkyl group substituted with one or moreNR₂ groups, wherein R is hydrogen or a C₁₋₆alkyl group defined above,wherein each C₁₋₆alkyl group can be optionally further substituted.

An “alkanesulfonylamino” group is a —NR—SO₂—C₁₋₆alkyl group, wherein Ris hydrogen or a C₁₋₆alkyl group defined above, wherein each C₁₋₆alkylgroup can be optionally substituted.

A “C₃₋₁₀cycloalkyl” group is a cyclic alkyl group of from 3 to 10 carbonatoms having a single cyclic ring or multiple condensed or bridged ringswhich can be optionally substituted with from 1 to 3 alkyl groups. Suchcycloalkyl groups include, by way of example, single ring structuressuch as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl,2-methylcyclooctyl, and the like, or multiple or bridged ring structuressuch as adamantanyl and the like. A —(C₃₋₁₀cycloalkyl) group can besubstituted or unsubstituted. Such substituted cycloalkyl groupsinclude, by way of example, cyclohexanone and the like.

A “carboxyl” or “carboxy” is a —COOH group.

A “halogen” is fluorine, chlorine, bromine or iodine.

An “aryl” group is an unsaturated aromatic carbocyclic group of from 6to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl). Particular aryls includephenyl, biphenyl, naphthyl and the like. An aryl group can besubstituted or unsubstituted.

A “C₃₋₁₀heteroaryl” group is an aryl ring system having one to fourheteroatoms as ring atoms in a heteroaromatic ring system, wherein theremainder of the atoms are carbon atoms. Suitable heteroatoms includeoxygen, sulfur and nitrogen. In certain embodiments, the heterocyclicring system is monocyclic or bicyclic. Non-limiting examples include thefollowing:

wherein Q is CH₂, C═CH₂, O, S or NH. A —(C₃₋₁₀heteroaryl) group can besubstituted or unsubstituted.

A “C₃₋₁₀heterocycle” is an aromatic or non-aromatic cycloalkyl havingfrom 3 to 10 ring atoms in which one to four of the ring carbon atomsare independently replaced with a heteroatom from the group consistingof O, S and N. Representative examples of a heterocycle include, but arenot limited to, azetidine, benzofuranyl, benzothiophene, indolyl,benzopyrazolyl, coumarinyl, isoquinolinyl, morpholinyl, pyrrolyl,pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl,triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl,pyridazinyl, isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane,4,5-dihydro-1H-imidazolyl, tetrahydropyran, tetrahydrofuran andtetrazolyl. Additional non-limiting examples include the following:

including stereoisomers and enantiomers thereof,

wherein each occurrence of X is independently CH₂, O, S or N and R⁴ isH, substituted or unsubstituted C₁₋₆alkyl, substituted or unsubstitutedaryl, substituted or unsubstituted C₃₋₁₀cycloalkyl, substituted orunsubstituted C₃₋₁₀heterocycle or substituted or unsubstitutedC₃₋₁₀heteroaryl. A —(C₃₋₁₀heteroaryl) group can be substituted orunsubstituted. A —(C₃₋₁₀heterocycle) group can be substituted orunsubstituted.

A “heterocyclocarbonyl” group is a —C(O)—C₃₋₁₀heterocycle group, whereinC₃₋₁₀heterocycle is as described herein, wherein the C₃₋₁₀heterocyclegroup can be optionally substituted.

A “hydroxyalkyl” group is an alkyl group as described above substitutedwith one or more hydroxy groups.

In one embodiment, when the groups described herein are said to be“substituted,” they may be substituted with any substituent orsubstituents that do not adversely affect the activity of theAminopurine Compound. Examples of substituents are those found in theexemplary compounds and embodiments disclosed herein, as well as halogen(chloro, iodo, bromo, or fluoro); C₁₋₆ alkyl; C₂₋₆ alkenyl; C₂₋₆alkynyl; hydroxyl; C₁₋₆ alkoxyl; amino; nitro; thiol; thioether; imine;cyano; amido; phosphonato; phosphine; carboxyl; thiocarbonyl; sulfonyl;sulfonamide; ketone; aldehyde; ester; oxygen (═O); haloalkyl (e.g.,trifluoromethyl); B(OH)₂, carbocyclic cycloalkyl, which may bemonocyclic or fused or non-fused polycyclic (e.g., cyclopropyl,cyclobutyl, cyclopentyl, or cyclohexyl), or a heterocycloalkyl, whichmay be monocyclic or fused or non-fused polycyclic (e.g., pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, or thiazinyl); carbocyclic orheterocyclic, monocyclic or fused or non-fused polycyclic aryl (e.g.,phenyl, naphthyl, pyrrolyl, indolyl, furanyl, thiophenyl, imidazolyl,oxazolyl, isoxazolyl, thiazolyl, triazolyl, tetrazolyl, pyrazolyl,pyridinyl, quinolinyl, isoquinolinyl, acridinyl, pyrazinyl, pyridazinyl,pyrimidinyl, benzimidazolyl, benzothiophenyl, or benzofuranyl); amino(primary, secondary, or tertiary); O-lower alkyl; O-aryl, aryl;aryl-lower alkyl; CO₂CH₃; CONH₂; OCH₂CONH₂; NH₂; SO₂NH₂; OCHF₂; CF₃;OCF₃.

“JNK” means a protein or an isoform thereof expressed by a JNK 1, JNK 2,or JNK 3 gene (Gupta, S., Barrett, T., Whitmarsh, A. J., Cavanagh, J.,Sluss, H. K., Derijard, B. and Davis, R. J. The EMBO J. 15:2760-2770(1996)).

As used herein, the term “pharmaceutically acceptable salt(s)” refers toa salt prepared from a pharmaceutically acceptable non-toxic acid orbase including an inorganic acid and base and an organic acid and base.Suitable pharmaceutically acceptable base addition salts of theAminopurine Compounds include, but are not limited to metallic saltsmade from aluminum, calcium, lithium, magnesium, potassium, sodium andzinc or organic salts made from lysine, N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine,meglumine(N-methylglucamine) and procaine. Suitable non-toxic acidsinclude, but are not limited to, inorganic and organic acids such asacetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic,citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic,glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic,lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic,succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonicacid. Specific non-toxic acids include hydrochloric, hydrobromic,phosphoric, sulfuric, and methanesulfonic acids. Examples of specificsalts thus include hydrochloride and mesylate salts. Others arewell-known in the art, see for example, Remington's PharmaceuticalSciences, 18^(th) eds., Mack Publishing, Easton Pa. (1990) or Remington:The Science and Practice of Pharmacy, 19^(th) eds., Mack Publishing,Easton Pa. (1995).

As used herein, the term “polymorph(s)” and related terms herein referto solid forms of the Aminopurine Compounds having different physicalproperties as a result of the order of the molecules in the crystallattice. The differences in physical properties exhibited by solid formsaffect pharmaceutical parameters such as storage stability,compressibility and density (important in formulation and productmanufacturing), and dissolution rates (an important factor indetermining bioavailability). Differences in stability can result fromchanges in chemical reactivity (e.g., differential oxidation, such thata dosage form discolors more rapidly when comprised of one solid formthan when comprised of another solid form) or mechanical changes (e.g.,tablets crumble on storage as a kinetically favored polymorph convertsto thermodynamically more stable solid form) or both (e.g., tablets ofone solid form are more susceptible to breakdown at high humidity). As aresult of solubility/dissolution differences, in the extreme case, somesolid form transitions may result in lack of potency or, at the otherextreme, toxicity. In addition, the physical properties of the crystalmay be important in processing, for example, one solid form might bemore likely to form solvates or might be difficult to filter and washfree of impurities (i.e., particle shape and size distribution might bedifferent between one solid form relative to the other).

As used herein and unless otherwise indicated, the term “clathrate”means an Aminopurine Compound, or a salt thereof, in the form of acrystal lattice that contains spaces (e.g., channels) that have a guestmolecule (e.g., a solvent or water) trapped within or a crystal latticewherein an Aminopurine Compound is a guest molecule.

As used herein and unless otherwise indicated, the term “hydrate” meansan Aminopurine Compound, or a salt thereof, that further includes astoichiometric or non-stoichiometric amount of water bound bynon-covalent intermolecular forces.

As used herein and unless otherwise indicated, the term “solvate” meansan Aminopurine Compound, or a salt thereof, that further includes astoichiometric or non-stoichiometric amount of a solvent bound bynon-covalent intermolecular forces.

As used herein and unless otherwise indicated, the term “prodrug” meansan Aminopurine Compound derivative that can hydrolyze, oxidize, orotherwise react under biological conditions (in vitro or in vivo) toprovide an active compound, particularly an Aminopurine Compound.Examples of prodrugs include, but are not limited to, derivatives andmetabolites of an Aminopurine Compound that include biohydrolyzablemoieties such as biohydrolyzable amides, biohydrolyzable esters,biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzableureides, and biohydrolyzable phosphate analogues. In certainembodiments, prodrugs of compounds with carboxyl functional groups arethe lower alkyl esters of the carboxylic acid. The carboxylate estersare conveniently formed by esterifying any of the carboxylic acidmoieties present on the molecule. Prodrugs can typically be preparedusing well-known methods, such as those described by Burger's MedicinalChemistry and Drug Discovery 6^(th) ed. (Donald J. Abraham ed., 2001,Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985,Harwood Academic Publishers Gmfh).

As used herein and unless otherwise indicated, the term “stereoisomer”or “stereomerically pure” means one stereoisomer of an AminopurineCompound that is substantially free of other stereoisomers of thatcompound. For example, a stereomerically pure compound having one chiralcenter will be substantially free of the opposite enantiomer of thecompound. A stereomerically pure compound having two chiral centers willbe substantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, greater than about 90% by weight ofone stereoisomer of the compound and less than about 10% by weight ofthe other stereoisomers of the compound, greater than about 95% byweight of one stereoisomer of the compound and less than about 5% byweight of the other stereoisomers of the compound, or greater than about97% by weight of one stereoisomer of the compound and less than about 3%by weight of the other stereoisomers of the compound. The AminopurineCompounds can have chiral centers and can occur as racemates, individualenantiomers or diastereomers, and mixtures thereof. All such isomericforms are included within the embodiments disclosed herein, includingmixtures thereof.

Various Aminopurine Compounds contain one or more chiral centers, andcan exist as racemic mixtures of enantiomers, mixtures of diastereomersor enantiomerically or optically pure compounds. The use ofstereomerically pure forms of such Aminopurine Compounds, as well as theuse of mixtures of those forms are encompassed by the embodimentsdisclosed herein. For example, mixtures comprising equal or unequalamounts of the enantiomers of a particular Aminopurine Compound may beused in methods and compositions disclosed herein. These isomers may beasymmetrically synthesized or resolved using standard techniques such aschiral columns or chiral resolving agents. See, e.g., Jacques, J., etal., Enantiomers, Racemates and Resolutions (Wiley-Interscience, NewYork, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); andWilen, S. H., Tables of Resolving Agents and Optical Resolutions p. 268(E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972).

It should also be noted the Aminopurine Compounds include E and Zisomers, or a mixture thereof, and cis and trans isomers or a mixturethereof. In certain embodiments, the Aminopurine Compounds are isolatedas either the E or Z isomer. In other embodiments, the AminopurineCompounds are a mixture of the E and Z isomers.

The term “effective amount” in connection with an Aminopurine Compoundcan mean an amount capable of treating or preventing a disease disclosedherein, such as cancer, a cardiovascular disease, a renal disease, anautoimmune condition, an inflammatory condition, macular degeneration,ischemia-reperfusion injury, pain and related syndromes, disease-relatedwasting, an asbestos-related condition, pulmonary hypertension, centralnervous system (CNS) injury/damage or a condition treatable orpreventable by inhibition of a kinase pathway, in one embodiment, theJNK pathway.

As used herein, the term “macular degeneration” encompasses all forms ofmacular degenerative diseases regardless of a patient's age, althoughsome macular degenerative diseases are more common in certain agegroups. These include, but are not limited to, Best's disease orvitelliform (most common in patients under about seven years of age);Stargardt's disease, juvenile macular dystrophy or fundus flavimaculatus(most common in patients between about five and about 20 years of age);Behr's disease, Sorsby's disease, Doyne's disease or honeycomb dystrophy(most common in patients between about 30 and about 50 years of age);and age-related macular degeneration (most common in patients of about60 years of age or older). In one embodiment, the cause of the maculardegenerative disease is genetic. In another embodiment, the cause of themacular degenerative disease is physical trauma. In another embodiment,the cause of the macular degenerative disease is diabetes. In anotherembodiment, the cause of the macular degenerative disease ismalnutrition. In another embodiment, the cause of the maculardegenerative disease is infection.

As used herein, the phrase “ischemia-reperfusion injury” includes injurythat occurs during or as a result of surgery, including, but not limitedto, coronary artery bypass graft surgery, percutaneous transluminalcoronary angioplasty, orthopedic surgery, organ/vessel surgery,plaque/tumor removal surgery or organ/tissue transplant surgery (donoror recipient). The phrase “ischemia-reperfusion injury” also includesinjury that occurs to an organ or tissue ex vivo prior to transplant.

As used herein, the phrase “pain and related syndromes” includesnociceptive pain, such as that resulting from physical trauma (e.g., acut or contusion of the skin; or a chemical or thermal burn),osteoarthritis, rheumatoid arthritis or tendonitis; myofascial pain;neuropathic pain, such as that associated with stroke, diabeticneuropathy, luetic neuropathy, postherpetic neuralgia, trigeminalneuralgia, fibromyalgia, or painful neuropathy induced iatrogenically bydrugs such as vincristine, velcade or thalidomide; or mixed pain (i.e.,pain with both nociceptive and neuropathic components). Further types ofpain that can be treated or prevented by administering an effectiveamount of an Aminopurine Compound to a patient in need thereof include,but are not limited to, visceral pain; headache pain (e.g., migraineheadache pain); CRPS; CRPS type I; CRPS type II; RSD; reflexneurovascular dystrophy; reflex dystrophy; sympathetically maintainedpain syndrome; causalgia; Sudeck atrophy of bone; algoneurodystrophy;shoulder hand syndrome; post-traumatic dystrophy; autonomic dysfunction;cancer-related pain; phantom limb pain; chronic fatigue syndrome;post-operative pain; spinal cord injury pain; central post-stroke pain;radiculopathy; sensitivity to temperature, light touch or color changeto the skin (allodynia); pain from hyperthermic or hypothermicconditions; and other painful conditions (e.g., diabetic neuropathy,luetic neuropathy, postherpetic neuralgia, trigeminal neuralgia).

The term “disease-related wasting” means wasting (e.g., a loss ofphysical bulk through the breakdown of bodily tissue) associated with adisease such as HIV, AIDS, cancer, end-stage renal disease, kidneyfailure, chronic heart disease, obstructive pulmonary disease,tuberculosis, rheumatoid arthritis, a chronic inflammatory disease(e.g., scleroderma or mixed connective tissue disease) or a chronicinfectious disease (e.g., osteoarthritis or bacterial endocarditis).

The term “asbestos-related disease” includes diseases and disorders suchas malignant mesothelioma, asbestosis, malignant pleural effusion,benign pleural effusion, pleural plaque, pleural calcification, diffusepleural thickening, round atelectasis, and bronchogenic carcinoma, aswell as symptoms of asbestos-related diseases and disorders such asdyspnea, obliteration of the diaphragm, radiolucent sheet-likeencasement of the pleura, pleural effusion, pleural thickening,decreased size of the chest, chest discomfort, chest pain, easyfatigability, fever, sweats and weight loss.

The term “pulmonary hypertension” includes diseases characterized bysustained elevations of pulmonary artery pressure as well as symptomsassociated with pulmonary hypertension such as dyspnea, fatigue,weakness, chest pain, recurrent syncope, seizures, light-headedness,neurologic deficits, leg edema and palpitations.

The term “central nervous system (CNS) injury/damage” includes, but isnot limited to, primary brain injury, secondary brain injury, traumaticbrain injury, focal brain injury, diffuse axonal injury, head injury,concussion, post-concussion syndrome, cerebral contusion and laceration,subdural hematoma, epidermal hematoma, post-traumatic epilepsy, chronicvegetative state, complete SCI, incomplete SCI, acute SCI, subacute SCI,chronic SCI, central cord syndrome, Brown-Sequard syndrome, anteriorcord syndrome, conus medullaris syndrome, cauda equina syndrome,neurogenic shock, spinal shock, altered level of consciousness,headache, nausea, emesis, memory loss, dizziness, diplopia, blurredvision, emotional liability, sleep disturbances, irritability, inabilityto concentrate, nervousness, behavioral impairment, cognitive deficit,and seizure.

The term “patient” includes an animal, including, but not limited to, ananimal such a cow, monkey, horse, sheep, pig, chicken, turkey, quail,cat, dog, mouse, rat, rabbit or guinea pig, in one embodiment a mammal,in another embodiment a human.

4.2 Aminopurine Compounds

Provided herein are Aminopurine Compounds having the following formula(I):

and pharmaceutically acceptable salts, polymorphs, clathrates, solvates,hydrates, stereoisomers, enantiomers and prodrugs thereof,

wherein:

R¹ is substituted or unsubstituted C₁₋₆alkyl, substituted orunsubstituted aryl, substituted or unsubstituted C₃₋₁₀cycloalkyl,substituted or unsubstituted C₃₋₁₀heterocycle or substituted orunsubstituted C₃₋₁₀heteroaryl;

R² is H, substituted or unsubstituted C₁₋₆alkyl, substituted orunsubstituted aryl, substituted or unsubstituted C₃₋₁₀cycloalkyl,substituted or unsubstituted C₃₋₁₀heterocycle or substituted orunsubstituted C₃₋₁₀heteroaryl; and

R³ is aryl substituted with one or more halogens or C₃₋₁₀heteroarylsubstituted with one or more halogens, wherein the aryl orC₃₋₁₀heteroaryl group is optionally further substituted with one or moreC₁₋₆alkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino,alkylamino, carboxy, aminocarbonyl, cyano, acylamino,alkanesulfonylamino, tetrazolyl, triazolyl or imidazolyl groups.

In one embodiment, the Aminopurine Compounds of formula (I) are thosewherein R¹ is phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is substituted phenyl, in one embodiment alkoxysubstituted phenyl, in one embodiment p-alkoxy substituted phenyl, andin one embodiment p-methoxy substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is m-alkoxy substituted phenyl, in one embodimentm-methoxy substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is trifluoromethyl substituted phenyl, in oneembodiment p-trifluoromethyl substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is C₁₋₆alkyl, in one embodiment isopropyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is p-halo substituted phenyl, in one embodimentp-fluoro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is p-C₁₋₆alkyl substituted phenyl, in one embodimentp-methyl substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is o-halo substituted phenyl, in one embodimento-fluoro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is m,p-dihalo substituted phenyl, in one embodimentm,p-dichloro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is m-cyano substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is p-C₃₋₁₀heterocycle substituted phenyl, in oneembodiment p-morpholino substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is p-sulfonyl substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is C₃₋₁₀heteroaryl, in one embodiment pyridine orpyridinone.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is C₃₋₁₀heterocycle, in one embodiment piperidine,piperidin-2-one, pyrrolidinone or tetrahydropyran.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is N-substituted piperidine, in one embodimentN-sulfonyl substituted piperidine.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is C₃₋₁₀cycloalkyl, in one embodiment cyclohexyl,cyclopentyl or cyclopropyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is substituted C₃₋₁₀cycloalkyl, in one embodimentC₃₋₁₀cycloalkyl substituted with one or more C₁₋₆alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy,heterocyclocarbonyl, aminocarbonyl, cyano, acylamino,alkanesulfonylamino, tetrazolyl, triazolyl or imidazolyl groups.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is substituted C₃₋₁₀cycloalkyl, in one embodimentC₃₋₁₀cycloalkyl substituted with one or more alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, amino, aminoalkyl, amido, amidoalkyl,carboxy, heterocyclocarbonyl, sulfonamide or sulfonaminoalkyl groups.Cyclohexyl and cyclopentyl are particular C₃₋₁₀cycloalkyl groups.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is cyclohexyl substituted with one or more alkyl,hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, aminoalkyl, amido,amidoalkyl, carboxy, heterocyclocarbonyl, sulfonamide orsulfonaminoalkyl groups.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is C₁₋₆alkyl, in one embodiment methyl, ethyl, propyl(e.g., n-propyl or isopropyl) or butyl (e.g., isobutyl).

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is substituted C₁₋₆alkyl, in one embodiment phenyl,hydroxy, C₃₋₁₀cycloalkyl, or oxirane substituted C₁₋₆alkyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is benzyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R¹ is substituted C₁₋₆alkyl, in one embodimentC₃₋₁₀heterocycle (e.g., piperidine or pyrrolidine substituted C₁₋₆alkyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is substituted or unsubstituted C₁₋₆alkyl, substitutedor unsubstituted aryl, substituted or unsubstituted C₃₋₁₀heterocycle orsubstituted or unsubstituted C₃₋₁₀heteroaryl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is substituted or unsubstituted C₃₋₁₀cycloalkyl, in oneembodiment cyclohexyl, cyclopentyl, cyclobutyl or cyclopropyl.Cyclohexyl and cyclopentyl are specific C₃₋₁₀cycloalkyl groups. In oneembodiment, C₃₋₁₀cycloalkyl substitutents include C₁₋₆alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, amino, aminoalkyl, amido, amidoalkyl,carboxy, heterocyclocarbonyl, sulfonamide and sulfonaminoalkyl groups.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is cyclohexyl or cyclopentyl substituted with one ormore C₁₋₆alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, amino,aminoalkyl, amido, amidoalkyl, carboxy, heterocyclocarbonyl, sulfonamideor sulfonaminoalkyl groups.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is cyclohexyl or cyclopentyl substituted with one ormore C₁₋₆alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, amino,alkylamino, carboxy, heterocyclocarbonyl, aminocarbonyl, cyano,acylamino, alkanesulfonylamino, tetrazolyl, triazolyl or imidazolylgroups.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is C₁₋₆alkyl, in one embodiment butyl (e.g., n-butyl,isobutyl or t-butyl), propyl (e.g., isopropyl), ethyl or methyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is substituted C₁₋₆alkyl, in one embodiment cyano,C₃₋₁₀cycloalkyl or hydroxy substituted C₁₋₆alkyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is substituted C₁₋₆alkyl, in one embodimentC₃₋₁₀heterocycle (e.g., piperidine or pyrrolidine) hydroxy or amidosubstituted C₁₋₆alkyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is aryl, in one embodiment phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is C₃₋₁₀heterocycle, in one embodiment piperidine,piperidin-2-one, tetrahydropyran, tetrahydrofuran or azetidine.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is C₃₋₁₀heterocycle, in one embodiment a sulfurcontaining C₃₋₁₀heterocycle, including but not limited to4-(1,1-dioxo)thiopyranyl and 3-(1,1-dioxo)thiofuranyl. In a particularembodiment, R² is a sulfur, sulfonyl or sulfonamido containingC₃₋₁₀heterocycle.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is substituted C₃₋₁₀heterocycle, in one embodimentacetyl substituted piperidine.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is substituted or unsubstituted 3-oxetanyl,3-tetrahydrofuranyl, 4-tetrahydropyranyl, 4-piperidinyl,4-(1-acy)-piperidinyl, 4-(1-alkanesulfonyl)piperidinyl, 3-pyrrolidinyl,3-(1-acyl)pyrrolidinyl or 3-(1-alkanesulfonyl)pyrrolidinyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is o-halo substituted phenyl, in one embodimento-fluoro or chloro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is m-halo substituted phenyl, in one embodimentm-fluoro or chloro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is p-halo substituted phenyl, in one embodimentp-fluoro or chloro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is m,p-dihalo substituted phenyl, in one embodimentm,p-difluoro or dichloro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is o,m-dihalo substituted phenyl, in one embodimento,m-difluoro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is o,p-dihalo substituted phenyl, in one embodimento,p-difluoro substituted phenyl, o-fluoro-p-bromo substituted phenyl oro-fluoro-p-chloro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is o,o-dihalo substituted phenyl, in one embodimento,o-difluoro substituted phenyl or o-chloro-o-fluoro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is 2,4,6-trihalo substituted phenyl, in one embodimenttrifluoro substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is o-halo substituted, in one embodiment o-fluoro orchloro substituted, and m-trifluoromethyl substituted phenyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is halo substituted C₃₋₁₀heteroaryl, in one embodimenthalo substituted pyridine.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is not aminoethyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is not a five-membered heterocyclic ring.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is not a five-membered N-containing heterocyclic ring.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is not a five-membered O-containing heterocyclic ring.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is not 2-tetrahydrofuranyl.

In another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R² is not 2-pyrrolidinyl.

In a further embodiment, provided herein are Aminopurine Compounds offormula (I), and pharmaceutically acceptable salts, polymorphs,clathrates, solvates, hydrates, stereoisomers and prodrugs thereof,wherein:

R¹ is substituted or unsubstituted C₁₋₆alkyl, substituted orunsubstituted aryl, substituted or unsubstituted C₃₋₁₀cycloalkyl,substituted or unsubstituted C₃₋₁₀heterocycle or substituted orunsubstituted C₃₋₁₀heteroaryl;

R³ is aryl or C₃₋₁₀heteroaryl, each being substituted with one or morehalogens;

X is at each occurrence independently CH₂, O, S or N;

R⁴ and R⁵ are at each occurrence independently H, substituted orunsubstituted C₁₋₆alkyl, substituted or unsubstituted aryl, substitutedor unsubstituted C₃₋₁₀cycloalkyl, substituted or unsubstitutedC₃₋₁₀heterocycle or substituted or unsubstituted C₃₋₁₀heteroaryl; or R⁴and R⁵ taken together with the N atom to which they are attached form asubstituted or unsubstituted 5-7 membered heterocycle; and

n is at each occurrence independently an integer ranging from 0 to 3.

In a another embodiment, the Aminopurine Compounds of formula (I) arethose wherein R³ is:

wherein:

X is at each occurrence independently F, Cl, Br or I;

R₆ is C₁₋₆alkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino,alkylamino, carboxy, aminocarbonyl, cyano, acylamino,alkanesulfonylamino, tetrazolyl, triazolyl or imidazolyl;

m is an integer ranging from 1 to 5; and

p is an integer ranging from 0 to 4.

In a further embodiment, p is an integer ranging from 1 to 4.

In a further embodiment, provided herein are Aminopurine Compoundshaving the following formula (II):

and pharmaceutically acceptable salts, polymorphs, clathrates, solvates,hydrates, stereoisomers, enantiomers and prodrugs thereof,

wherein:

X is at each occurrence independently F, Cl, Br or I;

R² is:

R⁴ and R⁵ are at each occurrence independently H, substituted orunsubstituted C₁₋₆alkyl, substituted or unsubstituted aryl, substitutedor unsubstituted C₃₋₁₀cycloalkyl, substituted or unsubstitutedC₃₋₁₀heterocycle or substituted or unsubstituted C₃₋₁₀heteroaryl; or R⁴and R⁵ taken together with the N atom to which they are attached form asubstituted or unsubstituted 5-7 membered heterocycle;

R⁶ is C₁₋₆alkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino,alkylamino, carboxy, aminocarbonyl, cyano, acylamino,alkanesulfonylamino, tetrazolyl, triazolyl or imidazolyl;

m is an integer ranging from 1 to 5;

n is at each occurrence independently an integer ranging from 0 to 3;and

p is an integer ranging from 0-4.

In one embodiment, the Aminopurine Compounds of formula (II) are thosewherein X is fluoro.

In another embodiment, the Aminopurine Compounds of formula (II) arethose wherein X is fluoro and m is 3.

In another embodiment, p is 0.

In another embodiment, p is an integer ranging from 1 to 4.

In a further embodiment, provided herein are Aminopurine Compoundshaving the following formula (III):

and pharmaceutically acceptable salts, polymorphs, clathrates, solvates,hydrates, stereoisomers, enantiomers and prodrugs thereof,

wherein:

X is at each occurrence independently F, Cl, Br or I;

m is an integer ranging from 1 to 5;

p is an integer ranging from 0-4;

R¹ is:

R₆ is C₁₋₆alkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino,alkylamino, carboxy, aminocarbonyl, cyano, acylamino,alkanesulfonylamino, tetrazolyl, triazolyl or imidazolyl.

In one embodiment, the Aminopurine Compounds of formula (III) are thosewherein X is fluoro.

In another embodiment, the Aminopurine Compounds of formula (III) arethose wherein X is fluoro and m is 3.

In another embodiment, p is 0.

In another embodiment, p is an integer ranging from 1 to 4.

In one embodiment, provided herein are Aminopurine Compounds having thefollowing formula (IV):

and pharmaceutically acceptable salts, polymorphs, clathrates, solvates,hydrates, stereoisomers, enantiomers and prodrugs thereof,

wherein:

R³ is:

The following HPLC methods were used to characterized the compounds ofTable 1, below.

Method A=5→70% acetonitrile/water (0.1% TFA) over 20 minutes.

Method B=20→100% acetonitrile/water (0.1% TFA) over 20 minutes.

Method C=5→50% acetonitrile/water (0.1% TFA) over 20 minutes.

Method D=0→75% acetonitrile/water (0.1% TFA) over 20 minutes.

Method E: 0-75% Acetonitrile/Water (0.1% Formic Acid) over 5 minutesthen hold at 75% Acetonitrile/Water (0.1% Formic Acid) for 2 minutes.

Method F: 10% Acetonitrile/Water (0.1% Formic Acid) for first two10-100% Acetonitrile/Water (0.1% Formic Acid) from 2 minutes to 25minutes.

Representative Aminopurine Compounds are set forth in Table 1, below.TABLE 1 M + 1 HPLC Compound (min/method)

419.1 (10.32/B)

419.6 (9.40/B)

396.2 (2.51/E)

353.41 (3.49/E)

403.7 (15.98/D)

419.1 (10.57/B)

419.4 (9.517/B)

427.2 (9.3/B)

433.5 (9.817/B)

393.3 (8.950/B)

365.4 (8.083/B)

379.5 (8.517/B)

351.1 (8.98/B)

449.5 (7.967/B)

434.4 (6.283/B)

327.3 (8.433/B)

341.2 (8.883/B)

355.3 (9.267/B)

395.4 (10.183/B)

389.3 (9.533/B)

437.2 (9.37/B)

435.2 (10.89/B)

435.2 (10.89/B)

435.2 (10.89/B)

390.42 (8.717/B)

393.1 (8.917/B)

407.5 (9.317/B)

407.5 (9.467/B)

409.4 (10.583/A)

437.2 (13.94/A)

469.2 (15.06/A)

411.35 (3.27/E)

404.45 (13.388/A)

407.5 (10.315/B)

395.2 (12.1/A)

423.4 (11.68/A)

385.4 (11.164/A)

425.4 (8.25/B)

497.2 (18.04/B)

441.3 (13.557/B)

409.2 (9.216/B)

423.4 (8.633/B)

398.49 (9.067/B)

423.4 (8.633/B)

398.49 (9.067/B)

437 (8.82/B)

457.3 (11.82/B)

448.3 (8.867/A)

424.5 (9.083/B)

407.4 (10.37/B)

409.3 (9.269/B)

385 (9.643/B)

449.4 (10.717/A)

447.4 (11.63/A)

401.1 (8.757/B)

417.4 (9.65/B)

383.4 (11.5/C)

397.2 (12.286/C)

385.1 (10.496/B)

437.1 (7.58/B)

453.22 (8.28/B)

487 (8.87/B)

435.4 (8.133/B)

453.2 (8.22/B)

487.1 (8.92/B)

433.2 (7.93/B)

448.3 (8.85/A)

410.6 (9.517/A)

490.5 (7.617/B)

452.3 (11.072/B)

397.4 (5.15/E)

476.4 (8.983/A)

438.6 (9.25/A)

419.4 (7.53/B)

526.5 (9.056/B)

488.4 (10.741/B)

477.3 (9.141/B)

463.5 (8.992/B)

453.3 (8.767/B)

491.5 (8.767/B)

410.4 (9.1/A)

448.4 (8.8/A)

476.7 (7.55/B)

398.4 (8.72/A)

384.2 (11.75/A)

463 (9.941/B)

329.15 (3.41/E)

355.25 (3.72/E)

335.15 (3.25/E)

315.4 (3.24/E)

329.4 (3.25/E)

343.25 (3.62/E)

414.4 (9.27/B)

361.4 (3.37/E)

504.5 (10.98/A)

427.1 (9.183/B)

540.6 (13.3/C)

369.45 (3.9/E)

462.3 (9.02/A)

377.4 (11.02/F)

403.4 (12.16/F)

411.4 (12.84/F)

391.4 (11.85/F)

405.4 (12.39/F)

425.4 (13.54/F)

383.4 (4.04/E)

418.4 (6.12/F)

432.5 (6.48/F)

331.4 (7.7/F)

345.4 (8.63/F)

426.2 (8.550/A)

518.6 (8.48/B)

532.6 (8.82/B)

533.5 (6.53/B)

504.6 (8.00/B)

397.4 (8.12/B)

432.1 (7.60/B)

437.4 (11.040/B)

381.05 (4.55/E)

357.4 (8.84/F)

371.4 (9.09/F)

371.15 (3.17/E)

405.4 (10.3/F)

448.1 (3.72/E)

462.55 (11.69/F)

474.5 (11.97/F)

482.5 (13.31/F)

476.6 (11.019/B)

481.3 (8.67/A)

343.4 (6.25/B)

448.6 (10.733/A)

508.3 (8.517/B)

489.56

342.4 (8.05/A)

396 (6.33/B)

426.1 (9.707/B)

488.5 (9.045/B)

415.3 (8.22/B)

367.4 (3.76/E)

399.5 (3.23/E)

419.45 (3.31/E)

363.35 (4.39/E)

355.4 (3.67/E)

385.4 (3.05/E)

413.5 (8.62/B)

385.1 (3.28/E)

384.2 (9.58/A)

452.5 (7.717/B)

381.5 (3.97/E)

369.5 (3.84/E)

385.4 (7.15/B)

403.3 (7.28/B)

402.1 (6.18/B)

382.4 (2.23/E)

496.1 (7.87/B)

506.5 (8.40/B)

495.4 (11.467/B)

482.5 (9.48/A)

500.4 (10.52/A)

384.5 (8.65/A)

444.3 (10.837/B)

476.5 (7.417/B)

401.1 (9.97/A)

419.2 (10.13/A)

423.3 (8.37/A)

441.3 (8.82/B)

445.4 (6.800/B)

499.5 (8.83/A)

437.4 (12.757/B)

468.4 (9.50/A)

486.5 (9.67/A)

413.2 (11.061/B)

427.2 (11.072/B)

429 (10.67/A)

559.2 (9.85/A)

577.5 (10.02/A)

429.4 (10.57/A)

447.4 (10.80/A)

425.4 (8.067/A)

455.1 (7.100/B)

425.3 (11.78/A)

467.4 (11.45/A)

441.5 (7.563/B)

518.5 (6.967/B)

540.3 (10.1/A)

474.3 (15.381/B)

445.4 (10.944/B)

428.4 (9.17/A)

529 (16.526/B)

473.4 (14.624/B)

473.4 (14.624/B)

463.4 (7.050/B)

428.4 (9.05/A)

446.4 (9.30/A)

426.4 (5.600/A)

455.4 (9.617/A)

468.5 (6.567/B)

486 (9.467/A)

483.5 (7.58/B)

441.3 (9.483/A)

441.5 (9.433/A)

469.3 (8.533/B)

487.5 (10.66/A)

505.5 (8.017/B)

443.4 (8.156/B)

457 (8.000/B)

551.6 (9.95/C)

390.2 (7.10/B)

408.4 (8.52/B)

534.4 (7.52/B)

459 (7.983/B)

516.3 (7.48/B)

429 (9.233/B)

579.5 (10.37/C)

542.4 (10.57/C)

477.5 (8.233/B)

472.5 (8.467/A)

542.3 (10.57/C)

489.3 (2.6/E)

341.45 (3.44/E)

395.15 (4.87/E))

391.1 (3.87/E)

392.4 (8.12/F)

396.5 (2.32/E)

404.45 (2.71/E)

391.4 (11.67/F)

405.2 (4.02/E)

404.4 (8.42/F)

439.1 (4.37/E)

422.15 (2.82/E)

431.5 (4.27/E)

412.1 (3.08/E)

446.15 (2.68/E)

365.4 (8.85/F)

410.15 (2.53/E)

375.35 (3.76/E)

359.15 (3.00/E)

397.1 (3.82/E)

431.5 (12.84/F)

373.1 (3.21/E)

345.4 (8.05/F)

418.1 (2.64/E)

411.4 (10.52/A)

370.1 (2.30/E)

359.4 (8.83/F)

417.2 (4.14/E)

384.2 (2.51/E)

405.2 (4.07/E)

379.4 (9.03/F)

357 (11.41/F)

400.1 (2.33/E)

425.15 (4.25/E)

369.5 (3.86/E)

432.2 (2.54/E)

414.2 (2.55/E)

385.4 (9.83/F)

389.45 (3.97/E)

419.15 (4.19/E)

423.05 (4.09/E)

426.9 (6.84/F)

403.4 (4.18/E)

384.2 (2.32/E)

434.15 (2.67/E)

438.05 (2.49/E)

404.15 (2.61/E)

378.4 (7.18/F)

599.6 (10.05/C)

393.5 (3.25/E)

508 (9.467/A)

508 (9.433/A)

526 (9.700/A)

511.6 (9.77/C)

454.5 (8.883/A)

472.5 (10.150/C)

565.8 (9.783/A)

537.7 (8.417/A)

565.6 (10.10/C)

679 (9.367/A)

442.5 (7.650/A)

581.6 (10.95/C)

583.5 (9.133/A)

612.4 (10.0/A)

612 (9.417/A)

584.3 (9.6/A)

697 (11.528/A)

458 (9.933/A)

569 (10.167/A)

623.5 (8.97/A)

569.7 (8.68/A)

570.5 (10.50/A)

583.8 (9.92/A)

587 (10.217/A)

569.8 (8.593/C)

679 (9.600/A)

601.8 (10.032/A)

540.3 (10.1/A)

626.7 (10.167/A)

613.5 (10.83/C)

611.8 (11.28/C)

568.5 (9.32/A)

646.3 (10.42/C)

680.7 (8.75/A)

598.7 (9.184/A)

611.5 (10.360/A)

583.5 (10.92/C)

661.5 (11.65/C)

622.7 (9.484/A)

568.5 (9.256/A)

500.4 (10.436/A)

583.5 (C)

623.8 (C)

556.4 (C)

583.7 (C)

554.6 (C)

555.5 (C)

598.5 (A)

560.5 (B)

500.4 (B)

514.5 (B)

526.6 (B)

487.1 (A)

487.1 (A)

485.6 (A)

429.4 (B)

421.4 (B)

429.1 (B)

458.9 (B)

459.5 (B)

431.3 (B)

417.6 (B)

435.3 (B)

417.3 (B)

401 (B)

445 (A)

414 (A)

463 (A)

445 (A)

414 (A)

473 (A)

500.5 (A)

473 (A)

491 (A)

491 (A)

473 (A)

473 (A)

487 (A)

487 (A)

487 (A)

487 (A)

505 (A)

505 (A)

463 (A)

463 (A)

546 (A)

530 (A)

547 (A)

547 (A)

520 (A)

560 (A)

520 (A)

546 (A)

448 (A)

448 (A)

403.5 (B)

459.6 (A)

403.5 (A)

501.6 (A)

471.6 (A)

479.4 (A)

479.5 (A)

493.5 (A)

493.5 (A)

437.4 (A)

405.5 (A)

437.4 (A)

463.5 (A)

479.4 (A)

455.3 (A)

479.4 (A)

463.5 (A)

448.3 (A)

476.3 (A)

476.4 (A)

448.3 (A)

411.3 (A)

429.3 (A)

467.5 (A)

467.5 (A)

445.5 (A)

422.3 (A)

422.3 (11.33/A)

412.4 (A)

406.5 (A)

406.5 (A)

546.3 (A)

546.3 (A)

560.5 (A)

560.5 (A)

544.4 (A)

560.4 (A)

550.5 (A)

550.5 (A)

477.3 (A)

544.4 (A)

560.5 (A)

560.5 (A)

377.1 (A)

377 (A)

519.4 (B)

488.5 (A)

492.5 (A)

418.5 (A)

531.4 (A)

504.5 (A)

502.5 (A)

461.9 (A)

490.1 (A)

518.3 (A)

518.3 (A)

476.5 (A)

500.4 (A)

526.5 (A)

540.5 (A)

449.5 (A)

449.3 (A)

449.3 (A)

367.3 (A)

473 (A)

473 (A)

491 (A)

487 (A)

487 (A)

505 (A)

403 (A)

431 (A)

431 (A)

433 (A)

518 (B)

465.1 (A)

534 (A)

504 (A)

478 (A)

532 (A)

490 (A)

516 (A)

490 (A)

516 (A)

451 (A)

548 (A)

477 (B)

522.5 (A)

540.5 (A)

522.1 (A)

419.5 (A)

431.5 (A)

431.5 (A)

431.5 (A)

431.5 (A)

449.4 (A)

449.4 (A)

472.5 (A)

472.5 (A)

419.5 (A)

437.4 (A)

472.5 (A)

472.6 (A)

490.5 (A)

490.3 (A)

375.3 (A)

375.3 (A)

417.5 (A)

417.5 (A)

375.3 (A)

375.2 (A)

417 (A)

417.5 (A)

491.5 (A)

491.5 (B)

476.4 (A)

476.4 (A)

503.3 (A)

526.3 (A)

434.1 (A)

569.7 (A)

375 (B)

375.3 (A)

393.1 (A)

457.1 (A)

501.5 (A)

501.4 (A)

476.5 (A)

445.5 (A)

463.4 (A)

445 (A)

466.1 (A)

445.3 (A)

448.4 (A)

448.4 (A)

516.3 (A)

516.3 (A)

560.5 (A)

546.5 (A)

461.5 (A)

375.8 (A)

376 (A)

376.1 (A)

375.8 (A)

479.3 (A)

445.3 (A)

408.1 (A)

408.4 (A)

497 (A)

497 (A)

461.4 (A)

461.4 (A)

417.6 (A)

417.6 (A)

486.6 (A)

448.4 (A)

488.4 (A)

456.4 (A)

442.4 (A)

506 (A)

506 (A)

532.3 (A)

532.3 (A)

476.5 (A)

434.4 (A)

518.6 (A)

544.4 (A)

558 (A)

514.6 (A)

514.7 (A)

505.3 (A)

500.5 (A)

470.6 (A)

The compounds of Table 1 were purified by HPLC using one of theconditions A-F described above. The mass spectrometry data (M+1 ion) foreach compound is also set forth.

Aminopurine Compounds set forth in Table 1 were tested in the JNKinhibitor assays described herein and were found to have activity as JNKinhibitors.

4.3 Methods for Making Aminopurine Compounds

The Aminopurine Compounds can be made using conventional organicsyntheses. By way of example and not limitation, an Aminopurine Compoundcan be prepared as outlined in Schemes 1 and 2 shown below as well as inExamples 5.1 to 5.53.

Solid-phase reactions can be performed in, for example, 250 mLpolypropylene bottles for large reactions (>50 mL) or in 20 mL frittedpolypropylene syringes (<20 mL). All solvents used for washings are HPLCgrade unless otherwise stated. Each wash cycle is carried out with 100mL of solvent for the large vessels or 10 mL of solvent for smallvessels over 3-5 minutes unless otherwise stated. The reactions areshaken using a Lab-Line Instruments Titer Plate Shaker.

Synthesis of (2-Chloro-5-nitropyrimidin-4-yl)-R²amines

N,N-diisopropylethylamine is slowly added to a solution of2,4-dichloro-5-nitropyrimidine in THF at −78° C. The desired R² amine isdissolved in THF and added dropwise to the reaction mixture at −78° C.The reaction is stirred for about 1 hour at −78° C. and then allowed toslowly warm to room temperature overnight. Dichloromethane is added andthe organics are washed with water (500 mL) followed by NaHCO₃ (sat.aq., 2×500 mL). The organics are dried (MgSO₄), filtered, and thesolvent is removed in vacuo to provide the crude(2-chloro-5-nitropyrimidin-4-yl)-R²amine. The crude products are usedwithout further purification.

Reductive Amination with R¹ Amines

A solution of R¹ amine and HCl (a 4 M solution in dioxane) in 5%AcOH/DMF is added to 4-(4-formyl-3-methoxyphenoxy)butyryl AM resin in,for example, a 250 mL polypropylene tube. The resin suspension isagitated on a shaker for about 3 h and sodium triacetoxyborohydride isadded. Following shaking for about 1 h with periodic venting, the resinis washed twice with 5% AcOH/DMF using, for example, a polypropylenegas-dispersion tube under vacuum to aspirate off the solvent. A secondsolution of R¹ amine is added followed by agitation for 1 h. Sodiumtriacetoxyborohydride is added and the suspension is shaken overnight atroom temperature with venting of the reaction vessel for about the first1 h. The reaction vessel is drained and the resin is washed with DMF(2×), 50% MeOH/DMF (2×), DMF (3×) and CH₂Cl₂ (4×). The resin is thensplit into five, for example, 20 mL fritted polypropylene syringes usinga suspension in DMF.

N-Arylation with (2-Chloro-5-nitrolpyrimidin-4-yl)-R²amine

A solution of crude (2-chloro-5-nitropyrimidin-4-yl)-R²amine andNAN-diisopropylethylamine in CH₂Cl₂ is added to each syringe containinga different resin-bound secondary R¹ amine. After shaking the mixtureovernight, the reaction solution is drained and the resin is washed withDMF (5×) and CH₂Cl₂ (7×).

Nitro Reduction

A solution of SnCl₂ dihydrate in nitrogen-purged DMF is prepared in, forexample, a graduated 1 L glass bottle. N,N-Diisopropylethylamine isadded, the volume is adjusted to 1 L with nitrogen saturated DMF, andthe solution is purged for about 30 min. with a gentle stream ofnitrogen. The SnCl₂ solution is added to each resin-bound5-nitropyrimidine in, for example, a 20 mL fritted polypropylenesyringe. The reactions are capped and shaken overnight. The reactionsolutions are expelled, the resin is washed with nitrogen-purged DMF(3×) and freshly prepared SnCl₂ solution is added. After shakingovernight, the reaction solutions are expelled and the resin is washedwith nitrogen-purged DMF (3×). Following a third treatment with SnCl₂solution overnight, the reaction solutions are expelled, the resin iswashed with DMF (3×) followed by alternating washes with 50% DMF/H₂O andDMF (3× each). This is followed by washing the resin with MeOH (2×), DMF(2×) and CH₂Cl₂ (7×). Each resin is split into four, for example, 20 mLfritted polypropylene syringes using a suspension in DMF.

Aminopurine Formation

The desired isothiocyanate is added to a suspension of each resin-bound5-aminopyrimidine in DMF and CH₂Cl₂. The 20 mL fritted polypropylenesyringes containing the resin suspension are capped and allowed to shakeovernight. The reaction solutions are expelled, followed by the additionof a solution of DIC in CH₂Cl₂. The reactions are allowed to shake forabout 4 days, the reaction solutions are expelled and the resin iswashed with DMF (5×) and CH₂Cl₂ (7×). The resulting resin-boundaminopurines are dried in vacuo.

Cleavage from Resin

A 50% v/v TFA/CH₂Cl₂ solution is added to the resin-bound aminopurinesin, for example, 20 mL fritted polypropylene syringes. The resultingresin suspensions are allowed to shake overnight, the reaction solutionsare collected and dried in vacuo. The residues are partitioned betweenEtOAc and saturated aq. Na₂CO₃. After further extracting with EtOAc (2×4mL) the organic layers are collected, passed through polyethylene fritsand dried in vacuo. The residues are dissolved in DMSO, passed through asilica plug and purified using preparative HPLC to provide the desiredaminopurine.

Illustrative examples of Schemes 1 and 2 are set forth in Examples 5.1to 5.14, below.

Pharmaceutically acceptable salts of the Aminopurine Compounds can beformed by conventional and known techniques, such as by reacting aAminopurine Compound with a suitable acid as disclosed above. Such saltsare typically formed in high yields at moderate temperatures, and oftenare prepared by merely isolating the compound from a suitable acidicwash in the final step of the synthesis. The salt-forming acid maydissolved in an appropriate organic solvent, or aqueous organic solvent,such as an alkanol, ketone or ester. On the other hand, if theAminopurine Compound is desired in the free base form, it may beisolated from a basic final wash step, according to known techniques.For example, a typical technique for preparing hydrochloride salt is todissolve the free base in a suitable solvent, and dry the solutionthoroughly, as over molecular sieves, before bubbling hydrogen chloridegas through it.

4.4 Methods of Use

The Aminopurine Compounds have utility as pharmaceuticals to heal orprevent disease in animals or humans. Further, the Aminopurine Compoundsare active against protein kinases including those involved in cancer,cardiovascular disease, inflammatory diseases, autoimmune diseases andmetabolic disorders. Accordingly, provided herein are many uses of theAminopurine Compounds, including the treatment or prevention of thosediseases set forth below. The methods provided herein comprise theadministration of an effective amount of an Aminopurine Compound to apatient in need thereof.

Representative autoimmune conditions that the Aminopurine Compounds areuseful for treating or preventing include, but are not limited to,rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, multiplesclerosis, lupus, inflammatory bowel disease, ulcerative colitis,Crohn's disease, myasthenia gravis, Grave's disease and diabetes (e.g.,Type I diabetes).

Representative inflammatory conditions that the Aminopurine Compoundsare useful for treating or preventing include, but are not limited to,asthma and allergic rhinitis, bronchitis, chronic obstructive pulmonarydisease, cystic fibrosis, inflammatory bowel disease, irritable bowelsyndrome, Crohn's disease, mucous colitis, ulcerative colitis, diabetes(e.g., Type I diabetes and Type II diabetes) and obesity.

Representative metabolic conditions that the Aminopurine Compounds areuseful for treating or preventing include, but are not limited to,obesity and diabetes (e.g., Type II diabetes).

In a particular embodiment, provided herein are methods for thetreatment or prevention of insulin resistance. In certain embodiments,provided herein are methods for the treatment or prevention of insulinresistance that leads to diabetes (e.g., Type II diabetes).

In another embodiment, provided herein are methods for the treatment orprevention of syndrome X or metabolic syndrome.

In another embodiment, provide herein are methods for the treatment orprevention of diabetes.

In another embodiment, provide herein are methods for the treatment orprevention of Type II diabetes, Type I diabetes, slow-onset Type Idiabetes, diabetes insipidus (e.g., neurogenic diabetes insipidus,nephrogenic diabetes insipidus, dipsogenic diabetes insipidus, orgestagenic diabetes insipidus), diabetes mellitus, gestational diabetesmellitus, polycystic ovarian syndrome, maturity-onset diabetes, juvenilediabetes, insulin-dependant diabetes, non-insulin dependant diabetes,malnutrition-related diabetes, ketosis-prone diabetes, pre-diabetes(e.g., imparied glucose metabolism), cystic fibrosis related diabetes,hemochromatosis and ketosis-resistant diabetes.

In another embodiment, provided herein are methods for the treatment orprevention of fibrotic diseases and disorders. In a particularembodiment, provided herein are methods for the treatment or preventionof idiopathic pulmonary fibrosis, myelofibrosis, hepatic fibrosis,steatofibrosis and steatohepatitis.

Representative cardiovascular diseases that the Aminopurine Compoundsare useful for treating or preventing include, but are not limited to,stroke, myocardial infarction or ischemic damage to the heart, lung,gut, kidney, liver, pancreas, spleen or brain.

Representative cardiovascular and renal diseases that an AminopurineCompound containing or coated stent or stent graft is useful fortreating or preventing include atherosclerosis and the treatment orprevention of restenosis after vascular intervention such asangioplasty.

In another embodiment, provided herein are methods for improvedprocessing of beta-islet cells (e.g., human) for transplantation.

In another embodiment, provided herein are methods for improvedculturing of beta-islet cells (e.g., human) for transplantation.

In another embodiment, provided herein are methods for improvedviability of beta-islet cells (e.g., human) for transplantation.

In another embodiment, provided herein are methods for improvedgraft-survival of beta-islet cells (e.g., human) for transplantation.

In another embodiment, provided herein are methods for improvedprocessing, culturing, viability and graft-survival of beta-islet cells(e.g., human) for transplantation.

An Aminopurine Compound containing or coated stent or stent graft canfurther comprise an effective amount of another active agent useful fortreating or preventing a cardiovascular or renal disease, including, butare not limited to, an anticoagulant agent, an antimetabolite agent, ananti-inflammatory agent, an antiplatelet agent, an antithrombin agent,an antimitotic agent, a cytostatic agent or an antiproliferative agent.

The Aminopurine Compounds are also useful for treating or preventingischemia/reperfusion injury in general. Accordingly, the AminopurineCompounds are useful for treating or preventing acute or chronic organtransplant rejection and for the preservation of tissue and organs.

Representative cancers that the Aminopurine Compounds are useful fortreating or preventing include, but are not limited to, cancers of thehead, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx,chest, bone, lung, colon, rectum, stomach, prostate, urinary bladder,uterine, cervix, breast, ovaries, testicles or other reproductiveorgans, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, andbrain or central nervous system.

Cancers within the scope of the methods provided herein include thoseassociated with BCR-ABL, and mutants or isoforms thereof, as well askinases from the src kinase family, kinases from the Rsk kinase family,kinases from the CDK family, kinases from the MAPK kinase family, andtyrosine kinases such as Fes, Lyn, and Syk kinases, and mutants orisoforms thereof.

In a particular embodiment, provided herein are methods for thetreatment or prevention of a disease or disorder associated with themodulation, for example inhibition, of a kinase, including, but are notlimited to, tyrosine-protein kinase (SYK), tyrosine-protein kinase(ZAP-70), protein tyrosine kinase 2 beta (PYK2), focal adhesion kinase 1(FAK), B lymphocyte kinase (BLK), hemopoietic cell kinase (HCK), v-yes-1Yamaguchi sarcoma viral related oncogene homolog (LYN), T cell-specificprotein-tyrosine kinase (LCK), proto-oncogene tyrosine-protein kinase(YES), proto-oncogene tyrosine-protein kinase (SRC), proto-oncogenetyrosine-protein kinase (FYN), proto-oncogene tyrosine-protein kinase(FGR), proto-oncogene tyrosine-protein kinase (FER), proto-oncogenetyrosine-protein kinase (FES), C-SRC kinase, protein-tyrosine kinase(CYL), tyrosine protein kinase (CSK), megakaryocyte-associatedtyrosine-protein kinase (CTK), tyrosine-protein kinase receptor (EPH),Ephrin type-A receptor 1, Ephrin type-A receptor 4 (EPHA4), Ephrintype-B receptor 3 (EPHB3), Ephrin type-A receptor 8 (EPHA8),neurotrophic tyrosine kinase receptor, type 1 (NTRK1), protein-tyrosinekinase (PTK2), syk-related tyrosine kinase (SRK), protein tyrosinekinase (CTK), tyro3 protein tyrosine kinase (TYRO3), brutonagammaglobulinemia tyrosine kinase (BTK), leukocyte tyrosine kinase(LTK), protein-tyrosine kinase (SYK), protein-tyrosine kinase (STY), tektyrosine kinase (TEK), elk-related tyro sine kinase (ERK), tyrosinekinase with immunoglobulin and egf factor homology domains (TIE),protein tyrosine kinase (TKF), neurotrophic tyrosine kinase, receptor,type 3 (NTRK3), mixed-lineage protein kinase-3 (MLK3), protein kinase,mitogen-activated 4 (PRKM4), protein kinase, mitogen-activated 1 (PRKM1), protein tyrosine kinase (PTK7), protein tyrosine kinase (EEK),minibrain (drosophila) homolog (MNBH), bone marrow kinase, x-linked(BMX), eph-like tyrosine kinase 1 (ETK1), macrophage stimulating Ireceptor (MST1R), btk-associated protein, 135 kd, lymphocyte-specificprotein tyrosine kinase (LCK), fibroblast growth factor receptor-2(FGFR2), protein tyrosine kinase-3 (TYK3), protein tyrosine kinase(TXK), tec protein tyrosine kinase (TEC), protein tyrosine kinase-2(TYK2), eph-related receptor tyrosine kinase ligand 1 (EPLG1), t-celltyrosine kinase (EMT), eph tyrosine kinase 1 (EPHT1), zona pellucidareceptor tyrosine kinase, 95 kd (ZRK), protein kinase,mitogen-activated, kinase 1 (PRKMK1), eph tyrosine kinase 3 (EPHT3),growth arrest-specific gene-6 (GAS6), kinase insert domain receptor(KDR), axl receptor tyrosine kinase (AXL), fibroblast growth factorreceptor-1 (FGFR1), v-erb-b2 avian erythroblastic leukemia viraloncogene homolog 2 (ERBB2), fms-like tyrosine kinase-3 (FLT3),neuroepithelial tyrosine kinase (NEP), neurotrophic tyrosine kinasereceptor-related 3 (NTRKR3), eph-related receptor tyrosine kinase ligand5 (EPLG5), neurotrophic tyrosine kinase, receptor, type 2 (NTRK2),receptor-like tyrosine kinase (RYK), tyrosine kinase, b-lymphocytespecific (BLK), eph tyrosine kinase 2 (EPHT2), eph-related receptortyrosine kinase ligand 2 (EPLG2), glycogen storage disease VIII,eph-related receptor tyrosine kinase ligand 7 (EPLG7), janus kinase 1(JAK1), fms-related tyrosine kinase-1 (FLT1), protein kinase,camp-dependent, regulatory, type I, alpha (PRKAR1A), wee-1 tyrosinekinase (WEE1), eph-like tyrosine kinase 2 (ETK2), receptor tyrosinekinase musk, insulin receptor (INSR), janus kinase 3 (JAK3), fms-relatedtyrosine kinase-3 ligand protein kinase c, beta 1 (PRKCB1), tyrosinekinase-type cell surface receptor (HER3), janus kinase 2 (JAK2), limdomain kinase 1 (LIMK1), dual specificity phosphatase 1 (DUSP1),hemopoietic cell kinase (HCK), tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein, eta polypeptide (YWHAH), retproto-oncogene (RET), tyrosine 3-monooxygenase/tryptophan5-monooxygenase activation protein, zeta polypeptide (YWHAZ), tyrosine3-monooxygenase/tryptophan 5-monooxygenase activation protein, betapolypeptide (YWHAB), hepatoma transmembrane kinase (HTK), map kinasekinase 6, phosphatidylinositol 3-kinase, catalytic, alpha polypeptide(PIK3CA), cyclin-dependent kinase inhibitor 3 (CDKN3), diacylglycerolkinase, delta, 130 kd, protein-tyrosine phosphatase, nonreceptor type,13 (PTPN13), abelson murine leukemia viral oncogene homolog 1 (ABL1),diacylglycerol kinase, alpha (DAGK1), focal adhesion kinase 2,epithelial discoidin domain receptor 1 (EDDR1), anaplastic lymphomakinase (ALK), phosphatidylinositol 3-kinase, catalytic, gammapolypeptide (PIK3CG), phosphatidylinositol 3-kinase regulatory subunit,(PIK3R1), eph homology kinase-1 (EHK1), v-kit hardy-zuckerman 4 felinesarcoma viral oncogene homolog (KIT), fibroblast growth factorreceptor-3 (FGFR3), vascular endothelial growth factor c (VEGFC),epidermal growth factor receptor (EGFR), oncogene (TRK), growth factorreceptor-bound protein-7 (GRB7), ras p21 protein activator (RASA2), metproto-oncogene (MET), src-like adapter (SLA), vascular endothelialgrowth factor (VEGF), vascular endothelial growth factor receptor(VEGFR), nerve growth factor receptor (NGFR), platelet derived growthfactor receptor (PDGFR), platelet derived growth factor receptor beta(PDGFRB), dual-specificity tyrosine-(Y)-phosphorylation regulated kinase2 (DYRK2), dual-specificity tyrosine-(Y)-phosphorylation regulatedkinase 3 (DYRK3), dual-specificity tyrosine-(Y)-phosphorylationregulated kinase 4 (DYRK4), dual-specificitytyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A),dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B(DYRK1B), CDC-like kinase 1 (CLK1), protein tyrosine kinase STY,CDC-like kinase 4 (CLK4), CDC-like kinase 2 (CLK2) or CDC-like kinase 3(CLK3).

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder associated with the modulation, forexample inhibition, of serine/threonine kinases or related molecules,including, but not limited to, cyclin-dependent kinase 7 (CDK7), racserine/threonine protein kinase, serine-threonine protein kinase n(PKN), serine/threonine protein kinase 2 (STK2), zipper protein kinase(ZPK), protein-tyrosine kinase (STY), bruton agammaglobulinemia tyrosinekinase (BTK), mkn28 kinase, protein kinase, x-linked (PRKX), elk-relatedtyrosine kinase (ERK), ribosomal protein s6 kinase, 90 kd, polypeptide 3(RPS6KA3), glycogen storage disease VIII, death-associated proteinkinase 1 (DAPK1), pctaire protein kinase 1 (PCTK1), protein kinase,interferon-inducible double-stranded ma (PRKR), activin a receptor, typeII-like kinase 1 (ACVRLK1), protein kinase, camp-dependent, catalytic,alpha (PRKACA), protein kinase, y-linked (PRKY), G protein-coupledreceptor kinase 2 (GPRK21), protein kinase c, theta form (PRKCQ), limdomain kinase 1 (LIMK1), phosphoglycerate kinase 1 PGK1), lim domainkinase 2 (LIMK2), c-jun kinase, activin a receptor, type II-like kinase2 (ACVRLK2), janus kinase 1 (JAK1), elk1 motif kinase (EMK1), male germcell-associated kinase (MAK), casein kinase 2, alpha-prime subunit(CSNK2A2), casein kinase 2, beta polypeptide (CSNK2B), casein kinase 2,alpha 1 polypeptide (CSNK2A1), ret proto-oncogene (RET), hematopoieticprogenitor kinase 1, conserved helix-loop-helix ubiquitous kinase(CHUK), casein kinase 1, delta (CSNK1D), casein kinase 1, epsilon(CSNK1E), v-akt murine thymoma viral oncogene homolog 1 (AKT1), tumorprotein p53 (TP53), protein phosphatase 1, regulatory (inhibitor)subunit 2 (PPP1R2), oncogene pim-1 (PIM1), transforming growthfactor-beta receptor, type II (TGFBR2), transforming growth factor-betareceptor, type I (TGFBR1), v-raf murine sarcoma viral oncogene homologb1 (BRAF), bone morphogenetic receptor type II (BMPR2), v-raf murinesarcoma 3611 viral oncogene homolog 1 (ARAF1), v-raf murine sarcoma 3611viral oncogene homolog 2 (ARAF2), protein kinase C (PKC), v-kithardy-zuckerman 4 feline sarcoma viral oncogene homolog (KIT) or c-KITreceptor (KITR).

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder associated with the modulation, forexample inhibition, of a MAP kinase, including, but not limited to,mitogen-activated protein kinase 3 (MAPK3), p44erk1, p44mapk,mitogen-activated protein kinase 3 (MAP kinase 3; p44), ERK1, PRKM3,P44ERK1, P44MAPK, mitogen-activated protein kinase 1 (MAPK1),mitogen-activated protein kinase kinase 1 (MEK1), MAP2K1protein tyrosinekinase ERK2, mitogen-activated protein kinase 2, extracellularsignal-regulated kinase 2, protein tyrosine kinase ERK2,mitogen-activated protein kinase 2, extracellular signal-regulatedkinase 2, ERK, p38, p40, p41, ERK2, ERT1, MAPK2, PRKM1, PRKM2, P42MAPK,p41mapk, mitogen-activated protein kinase 7 (MAPK7), BMK1 kinase,extracellular-signal-regulated kinase 5, BMK1, ERK4, ERK5, PRKM7,nemo-like kinase (NLK), likely ortholog of mouse nemo like kinase,mitogen-activated protein kinase 8 (MAPK8), protein kinase JNK1, JNK1beta protein kinase, JNK1 alpha protein kinase, c-Jun N-terminal kinase1, stress-activated protein kinase JNK1, JNK, JNK1, PRKM8, SAPK1,JNK1A2, JNK21B1/2, mitogen-activated protein kinase 10 (MAPK10), c-Junkinase 3, JNK3 alpha protein kinase, c-Jun N-terminal kinase 3, stressactivated protein kinase JNK3, stress activated protein kinase beta,mitogen-activated protein kinase 9 (MAPK9), MAP kinase 9, c-Jun kinase2, c-Jun N-terminal kinase 2, stress-activated protein kinase JNK2,JNK2, JNK2A, JNK2B, PRKM9, JNK-55, JNK2BETA, p54aSAPK, JNK2ALPHA,mitogen-activated protein kinase 14 (MAPK14), p38 MAP kinase, MAP kinaseMxi2, Csaids binding protein, MAX-interacting protein 2,stress-activated protein kinase 2A, p38 mitogen activated proteinkinase, cytokine suppressive anti-inflammatory drug binding protein, RK,p38, EXIP, Mxi2, CSBP1, CSBP2, CSPB1, PRKM14, PRKM15, SAPK2A, p38ALPHA,mitogen-activated protein kinase 11 (MAPK11), stress-activated proteinkinase-2, stress-activated protein kinase-2b, mitogen-activated proteinkinase p38-2, mitogen-activated protein kinase p38beta, P38B, SAPK2,p38-2, PRKM11, SAPK2B, p38Beta, P38BETA2, mitogen-activated proteinkinase 13 (MAPK13), stress-activated protein kinase 4, mitogen-activatedprotein kinase p38 delta, SAPK4, PRKM13, p38delta, mitogen-activatedprotein kinase 12 (MAPK12), p38gamma, stress-activated protein kinase 3,mitogen-activated protein kinase 3, ERK3, ERK6, SAPK3, PRKM12, SAPK-3,P38GAMMA, mitogen-activated protein kinase 6 (MAPK6), MAP kinase isoformp97, mitogen-activated 5 protein kinase, mitogen-activated 6 proteinkinase, extracellular signal-regulated kinase 3, extracellularsignal-regulated kinase, p97, ERK3, PRKM6, p97MAPK, mitogen-activatedprotein kinase 4 (MAPK4), Erk3-related protein kinase, mitogen-activated4 protein kinase (MAP kinase 4; p63), PRKM4, p63MAPK, ERK3-RELATED orExtracellular signal-regulated kinase 8 (ERK7).

More particularly, cancers and related disorders that can be treated orprevented by methods and compositions provided herein include but arenot limited to the following: Leukemias such as but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome (or a symptomthereof such as anemia, thrombocytopenia, neutropenia, bicytopenia orpancytopenia), refractory anemia (RA), RA with ringed sideroblasts(RARS), RA with excess blasts (RAEB), RAEB in transformation (RAEB-T),preleukemia and chronic myelomonocytic leukemia (CMML), chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as but not limited to Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as but notlimited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as but notlimited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma,malignant giant cell tumor, fibrosarcoma of bone, chordoma, periostealsarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma),fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma,lymphangiosarcoma, metastatic cancers, neurilemmoma, rhabdomyosarcoma,synovial sarcoma; brain tumors such as but not limited to, glioma,astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglialtumor, acoustic neurinoma, craniopharyngioma, medulloblastoma,meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breastcancer, including, but not limited to, adenocarcinoma, lobular (smallcell) carcinoma, intraductal carcinoma, medullary breast cancer,mucinous breast cancer, tubular breast cancer, papillary breast cancer,primary cancers, Paget's disease, and inflammatory breast cancer;adrenal cancer such as but not limited to pheochromocytom andadrenocortical carcinoma; thyroid cancer such as but not limited topapillary or follicular thyroid cancer, medullary thyroid cancer andanaplastic thyroid cancer; pancreatic cancer such as but not limited to,insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secretingtumor, and carcinoid or islet cell tumor; pituitary cancers such as butlimited to Cushing's disease, prolactin-secreting tumor, acromegaly, anddiabetes insipius; eye cancers such as but not limited to ocularmelanoma such as iris melanoma, choroidal melanoma, and cilliary bodymelanoma, and retinoblastoma; vaginal cancers such as squamous cellcarcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamouscell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,and Paget's disease; cervical cancers such as but not limited to,squamous cell carcinoma, and adenocarcinoma; uterine cancers such as butnot limited to endometrial carcinoma and uterine sarcoma; ovariancancers such as but not limited to, ovarian epithelial carcinoma,borderline tumor, germ cell tumor, and stromal tumor; esophageal cancerssuch as but not limited to, squamous cancer, adenocarcinoma, adenoidcyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma,sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell(small cell) carcinoma; stomach cancers such as but not limited to,adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading,diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma, gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to papillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer; testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, adenocarcinoma,leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers suchas but not limited to squamous cell carcinoma; basal cancers; salivarygland cancers such as but not limited to adenocarcinoma, mucoepidermoidcarcinoma, and adenoidcystic carcinoma; pharynx cancers such as but notlimited to squamous cell cancer, and verrucous; skin cancers such as butnot limited to, basal cell carcinoma, squamous cell carcinoma andmelanoma, superficial spreading melanoma, nodular melanoma, lentigomalignant melanoma, acral lentiginous melanoma; kidney cancers such asbut not limited to renal cell cancer, adenocarcinoma, hypemephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,Philadelphia and Murphy et al., 1997, Informed Decisions: The CompleteBook of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin,Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions provided herein are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal orignin, including fibrosarcoma and rhabdomyosarcoma; othertumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma andglioma; tumors of the central and peripheral nervous system, includingastrocytoma, glioblastoma multiforme, neuroblastoma, glioma, andschwannomas; solid and blood born tumors; tumors of mesenchymal origin,including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and othertumors, including melanoma, xenoderoma pegmentosum, keratoctanthoma,seminoma, thyroid follicular cancer and teratocarcinoma. It is alsocontemplated that cancers caused by aberrations in apoptosis would alsobe treated by the methods and compositions disclosed herein. Suchcancers may include but not be limited to follicular lymphomas,carcinomas with p53 mutations, hormone dependent tumors of the breast,prostate and ovary, and precancerous lesions such as familialadenomatous polyposis, and myelodysplastic syndromes. In specificembodiments, malignancy or dysproliferative changes (such as metaplasiasand dysplasias), or hyperproliferative disorders, are treated orprevented in the ovary, bladder, breast, colon, lung, skin, pancreas, oruterus. In other specific embodiments, sarcoma, melanoma, or leukemia istreated or prevented.

In another embodiment, the methods and compositions provided herein arealso useful for administration to patients in need of a bone marrowtransplant to treat a malignant disease (e.g., patients suffering fromacute lymphocytic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, myelodysplasticsyndrome (“preleukemia”), monosomy 7 syndrome, non-Hodgkin's lymphoma,neuroblastoma, brain tumors, multiple myeloma, testicular germ celltumors, breast cancer, lung cancer, ovarian cancer, melanoma, glioma,sarcoma or other solid tumors), those in need of a bone marrowtransplant to treat a non-malignant disease (e.g., patients sufferingfrom hematologic disorders, congenital immunodeficiencies,mucopolysaccharidoses, lipidoses, osteoporosis, Langerhan's cellhistiocytosis, Lesch-Nyhan syndrome or glycogen storage diseases), thoseundergoing chemotherapy or radiation therapy, those preparing to undergochemotherapy or radiation therapy and those who have previouslyundergone chemotherapy or radiation therapy.

In another embodiment, provided herein are methods for the treatment ofmyeloproliferative disorders or myelodysplastic syndromes, comprisingadministering to a patient in need thereof an effective amount of anAminopurine Compound or a composition thereof. In certain embodiments,the myeloproliferative disorder is polycythemia rubra vera; primarythrombocythemia; chronic myelogenous leukemia; acute or chronicgranulocytic leukemia; acute or chronic myelomonocytic leukemia;myelofibro-erythroleukemia; or agnogenic myeloid metaplasia.

In another embodiment, provided herein are methods for the treatment ofcancer or tumors resistant to other kinase inhibitors such as imatinibmesylate (STI-571 or Gleevec™) treatment, comprising administering to apatient in need thereof an effective amount of an Aminopurine Compoundor a composition thereof. In a particular embodiment, provided hereinare methods for the treatment of leukemias, including, but not limitedto, gastrointestinal stromal tumor (GIST), acute lymphocytic leukemia orchronic myelocytic leukemia resistant to imatinib mesylate (STI-571 orGleevec™) treatment, comprising administering to a patient in needthereof an effective amount of an Aminopurine Compound or a compositionthereof.

In a particular embodiment, provided herein are methods for thetreatment or prevention of airway hyperresponsiveness (AHR) and lunginflammation comprising administering an effective amount of anAminopurine Compound to a patient in need thereof.

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder caused, induced or exacerbated by anagonist including, but not limited to, ozone, cold or exercise,comprising administering an effective amount of an Aminopurine Compoundto a patient in need thereof. In a specific embodiment, provided hereinare methods for the treatment or prevention of asthma, bronchitis,rhinitis, COPD, lung inflammation and AHR caused, induced or exacerbatedby an agonist including, but not limited to, ozone, cold or exercise,comprising administering an effective amount of an Aminopurine Compoundto a patient in need thereof.

In another embodiment, provided herein are methods for the treatment orprevention of ozone-induced effects (e.g., adverse effects) on a lung,comprising administering an effective amount of an Aminopurine Compoundto a patient in need thereof.

In another embodiment, provided herein are methods for modulatinginflammatory cell recruitment and/or inflammatory gene expression in thelungs comprising administering an effective amount of an AminopurineCompound to a patient in need thereof.

In another embodiment, provided herein are methods for inhibitingneutrophil accumulation in bronchioalveolar lavage fluid comprisingadministering an effective amount of an Aminopurine Compound to apatient in need thereof.

In another embodiment, provided herein are methods for modulating (i.e.,inducing or inhibiting) expression of genes involved in oxidative stressresponse comprising administering an effective amount of an AminopurineCompound to a patient in need thereof.

In another embodiment, provided herein are methods for modulating (i.e.,inducing or inhibiting) expression of genes modulated by ozonecomprising administering an effective amount of an Aminopurine Compoundto a patient in need thereof. In a specific embodiment, provided hereinare methods for modulating (i.e., inducing or inhibiting) expression ofthe following genes comprising administering an effective amount of anAminopurine Compound to a patient in need thereof: Interleukin 6;Chemokine (C-X-C motif) ligand 1; A disintegrin-like and metalloprotease(reprolysin type) with thrombospondin type 1 motif, 4; Metallothionein1; Chemokine (C-X-C motif) ligand 2; Interleukin 1 receptor, type II;Small chemokine (C-C motif) ligand 11; Pentaxin related gene; Hemopexin;Matrix metalloproteinase 8; Tumor necrosis factor alpha induced protein6; Cyclin-dependent kinase inhibitor 1A (P21); FK506 binding protein 5;Protease, serine, 22; DNA-damage-inducible transcript 4; Similar tomPLZF(B)=promyelocytic leukemia zinc finger protein {alternativelyspliced} (LOC235320), mRNA; Cyclin-dependent kinase inhibitor 1A (P21);Double C2, beta; Suppressor of cytokine signaling 3; Metallothionein 2;Angiopoietin; Solute carrier family 27 (fatty acid transporter), member3; wingless-related MMTV integration site 7A; BB224790 RIKEN full-lengthenriched, adult male aorta and vein Mus musculus mRNA sequence;Calcitonin receptor-like; Glia maturation factor, beta; RIKENfull-length enriched library, clone:D230030K09; Mp78a12.x1Soares_thymus_(—)2NbMT Mus musculus cDNA clone IMAGE:575326 mRNAsequence; cDNA sequence BC025076; Interleukin 6 signal transducer; Myctarget 1; Tripartite motif protein 37; Frizzled homolog 2 (Drosophila);DNA segment, Chr 4, Wayne State University 53, expressed; Stromalantigen 2; HLA-B associated transcript 8; RIKEN cDNA 1110014P06 gene;GATA binding protein 3; AV218922 RIKEN full-length enriched, mRNAsequence; and PDZ domain containing, X chromosome.

In one embodiment, provided herein are methods for treating orpreventing a disease or disorder treatable or preventable by modulatinga kinase pathway, in one embodiment, the JNK pathway, comprisingadministering an effective amount of an Aminopurine Compound to apatient in need of the treating or preventing. Particular diseases whichare treatable or preventable by modulating, for example, inhibiting, akinase pathway, in one embodiment, the JNK pathway, include, but are notlimited to, rheumatoid arthritis; rheumatoid spondylitis;osteoarthritis; gout; asthma, bronchitis; allergic rhinitis; chronicobstructive pulmonary disease; cystic fibrosis; inflammatory boweldisease; irritable bowel syndrome; mucous colitis; ulcerative colitis;Crohn's disease; Huntington's disease; gastritis; esophagitis;hepatitis; pancreatitis; nephritis; multiple sclerosis; lupuserythematosus; Type II diabetes; obesity; atherosclerosis; restenosisfollowing angioplasty; left ventricular hypertrophy; myocardialinfarction; stroke; ischemic damages of heart, lung, gut, kidney, liver,pancreas, spleen and brain; acute or chronic organ transplant rejection;preservation of the organ for transplantation; organ failure or loss oflimb (e.g., including, but not limited to, that resulting fromischemia-reperfusion injury, trauma, gross bodily injury, car accident,crush injury or transplant failure); graft versus host disease;endotoxin shock; multiple organ failure; psoriasis; burn from exposureto fire, chemicals or radiation; eczema; dermatitis; skin graft;ischemia; ischemic conditions associated with surgery or traumaticinjury (e.g., vehicle accident, gunshot wound or limb crush); epilepsy;Alzheimer's disease; Parkinson's disease; immunological response tobacterial or viral infection; cachexia; angiogenic and proliferativediseases; solid tumor; and cancers of a variety of tissues such ascolon, rectum, prostate, liver, lung, bronchus, pancreas, brain, head,neck, stomach, skin, kidney, cervix, blood, larynx, esophagus, mouth,pharynx, urinary bladder, ovary or uterine.

4.5 Pharmaceutical Compositions and Routes of Administration

The Aminopurine Compounds can be administered to a patient orally orparenterally in the conventional form of preparations, such as capsules,microcapsules, tablets, granules, powder, troches, pills, suppositories,injections, suspensions and syrups. Suitable formulations can beprepared by methods commonly employed using conventional, organic orinorganic additives, such as an excipient (e.g., sucrose, starch,mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphateor calcium carbonate), a binder (e.g., cellulose, methylcellulose,hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone,gelatin, gum arabic, polyethyleneglycol, sucrose or starch), adisintegrator (e.g., starch, carboxymethylcellulose,hydroxypropylstarch, low substituted hydroxypropylcellulose, sodiumbicarbonate, calcium phosphate or calcium citrate), a lubricant (e.g.,magnesium stearate, light anhydrous silicic acid, talc or sodium laurylsulfate), a flavoring agent (e.g., citric acid, menthol, glycine ororange powder), a preservative (e.g., sodium benzoate, sodium bisulfite,methylparaben or propylparaben), a stabilizer (e.g., citric acid, sodiumcitrate or acetic acid), a suspending agent (e.g., methylcellulose,polyvinyl pyrroliclone or aluminum stearate), a dispersing agent (e.g.,hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax(e.g., cocoa butter, white petrolatum or polyethylene glycol). Theeffective amount of the Aminopurine Compounds in the pharmaceuticalcomposition may be at a level that will exercise the desired effect; forexample, about 0.005 mg/kg of a patient's body weight to about 10 mg/kgof a patient's body weight in unit dosage for both oral and parenteraladministration.

The dose of an Aminopurine Compound to be administered to a patient israther widely variable and can be subject to the judgment of ahealth-care practitioner. In general, the Aminopurine Compounds can beadministered one to four times a day in a dose of about 0.005 mg/kg of apatient's body weight to about 10 mg/kg of a patient's body weight in apatient, but the above dosage may be properly varied depending on theage, body weight and medical condition of the patient and the type ofadministration. In one embodiment, the dose is about 0.01 mg/kg of apatient's body weight to about 5 mg/kg of a patient's body weight, about0.05 mg/kg of a patient's body weight to about 1 mg/kg of a patient'sbody weight, about 0.1 mg/kg of a patient's body weight to about 0.75mg/kg of a patient's body weight or about 0.25 mg/kg of a patient's bodyweight to about 0.5 mg/kg of a patient's body weight. In one embodiment,one dose is given per day. In any given case, the amount of theAminopurine Compound administered will depend on such factors as thesolubility of the active component, the formulation used and the routeof administration.

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder comprising the administration ofabout 0.375 mg/day to about 750 mg/day, about 0.75 mg/day to about 375mg/day, about 3.75 mg/day to about 75 mg/day, about 7.5 mg/day to about55 mg/day or about 18 mg/day to about 37 mg/day of an AminopurineCompound to a patient in need thereof.

In another embodiment, provided herein are methods for the treatment orprevention of a disease or disorder comprising the administration ofabout 1 mg/day to about 1200 mg/day, about 10 mg/day to about 1200mg/day, about 100 mg/day to about 1200 mg/day, about 400 mg/day to about1200 mg/day, about 600 mg/day to about 1200 mg/day, about 400 mg/day toabout 800 mg/day or about 600 mg/day to about 800 mg/day of anAminopurine Compound to a patient in need thereof. In a particularembodiment, the methods disclosed herein comprise the administration of400 mg/day, 600 mg/day or 800 mg/day of an Aminopurine Compound to apatient in need thereof.

In another embodiment, provided herein are unit dosage formulations thatcomprise between about 1 mg and 200 mg, about 35 mg and about 1400 mg,about 125 mg and about 1000 mg, about 250 mg and about 1000 mg, or about500 mg and about 1000 mg of an Aminopurine Compound.

In a particular embodiment, provided herein are unit dosage formulationcomprising about 100 mg or 400 mg of an Aminopurine compound.

In another embodiment, provided herein are unit dosage formulations thatcomprise 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 35 mg, 50 mg, 70 mg,100 mg, 125 mg, 140 mg, 175 mg, 200 mg, 250 mg, 280 mg, 350 mg, 500 mg,560 mg, 700 mg, 750 mg, 1000 mg or 1400 mg of an Aminopurine Compound.

An Aminopurine Compound can be administered once, twice, three, four ormore times daily. In a particular embodiment, doses of 600 mg or lessare administered as a a once daily dose and doses of more than 600 mgare administered twice daily in an amount equal to one half of the totaldaily dose.

An Aminopurine Compound can be administered orally for reasons ofconvenience. In one embodiment, when administered orally, an AminopurineCompound is administered with a meal and water. In another embodiment,the Aminopurine Compound is dispersed in water or juice (e.g., applejuice or orange juice) and administered orally as a suspension.

The Aminopurine Compound can also be administered intradermally,intramuscularly, intraperitoneally, percutaneously, intravenously,subcutaneously, intranasally, epidurally, sublingually, intracerebrally,intravaginally, transdermally, rectally, mucosally, by inhalation, ortopically to the ears, nose, eyes, or skin. The mode of administrationis left to the discretion of the health-care practitioner, and candepend in-part upon the site of the medical condition.

In one embodiment, provided herein are capsules containing anAminopurine Compound without an additional carrier, excipient orvehicle.

In another embodiment, provided herein are compositions comprising aneffective amount of an Aminopurine Compound and a pharmaceuticallyacceptable carrier or vehicle, wherein a pharmaceutically acceptablecarrier or vehicle can comprise an excipient, diluent, or a mixturethereof. In one embodiment, the composition is a pharmaceuticalcomposition.

The compositions can be in the form of tablets, chewable tablets,capsules, solutions, parenteral solutions, troches, suppositories andsuspensions and the like. Compositions can be formulated to contain adaily dose, or a convenient fraction of a daily dose, in a dosage unit,which may be a single tablet or capsule or convenient volume of aliquid. In one embodiment, the solutions are prepared from water-solublesalts, such as the hydrochloride salt. In general, all of thecompositions are prepared according to known methods in pharmaceuticalchemistry. Capsules can be prepared by mixing an Aminopurine Compoundwith a suitable carrier or diluent and filling the proper amount of themixture in capsules. The usual carriers and diluents include, but arenot limited to, inert powdered substances such as starch of manydifferent kinds, powdered cellulose, especially crystalline andmicrocrystalline cellulose, sugars such as fructose, mannitol andsucrose, grain flours and similar edible powders.

Tablets can be prepared by direct compression, by wet granulation, or bydry granulation. Their formulations usually incorporate diluents,binders, lubricants and disintegrators as well as the compound. Typicaldiluents include, for example, various types of starch, lactose,mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such assodium chloride and powdered sugar. Powdered cellulose derivatives arealso useful. Typical tablet binders are substances such as starch,gelatin and sugars such as lactose, fructose, glucose and the like.Natural and synthetic gums are also convenient, including acacia,alginates, methylcellulose, polyvinylpyrrolidine and the like.Polyethylene glycol, ethylcellulose and waxes can also serve as binders.

A lubricant might be necessary in a tablet formulation to prevent thetablet and punches from sticking in the die. The lubricant can be chosenfrom such slippery solids as talc, magnesium and calcium stearate,stearic acid and hydrogenated vegetable oils. Tablet disintegrators aresubstances that swell when wetted to break up the tablet and release thecompound. They include starches, clays, celluloses, algins and gums.More particularly, corn and potato starches, methylcellulose, agar,bentonite, wood cellulose, powdered natural sponge, cation-exchangeresins, alginic acid, guar gum, citrus pulp and carboxymethyl cellulose,for example, can be used as well as sodium lauryl sulfate. Tablets canbe coated with sugar as a flavor and sealant, or with film-formingprotecting agents to modify the dissolution properties of the tablet.The compositions can also be formulated as chewable tablets, forexample, by using substances such as mannitol in the formulation.

When it is desired to administer an Aminopurine Compound as asuppository, typical bases can be used. Cocoa butter is a traditionalsuppository base, which can be modified by addition of waxes to raiseits melting point slightly. Water-miscible suppository bases comprising,particularly, polyethylene glycols of various molecular weights are inwide use.

The effect of the Aminopurine Compound can be delayed or prolonged byproper formulation. For example, a slowly soluble pellet of theAminopurine Compound can be prepared and incorporated in a tablet orcapsule, or as a slow-release implantable device. The technique alsoincludes making pellets of several different dissolution rates andfilling capsules with a mixture of the pellets. Tablets or capsules canbe coated with a film that resists dissolution for a predictable periodof time. Even the parenteral preparations can be made long-acting, bydissolving or suspending the Aminopurine Compound in oily or emulsifiedvehicles that allow it to disperse slowly in the serum.

5. EXAMPLES

The following Examples are presented by way of illustration, notlimitation.

Example 5.1 Synthesis of4-({8-[(2,6-Difluorophenyl)amino]-9-cyclopentylpurin-2-yl}amino)trans-cyclohexan-1-ol

1. (2-Chloro-5-nitropyrimidin-4-yl)cyclopentylamine

2,4-Dichloro-5-nitropyrimidine (10.31 mmol, 2 g) and cyclopentylamine(10.31 mmol, 1.02 mL) were dissolved in THF (60 mL) and cooled to −78°C. N,N-diisopropylethylamine (10.31 mmol, 1.8 mL) was added dropwise.The reaction mixture was stirred at −78° C. for about 45 minutes. Thecooling bath was removed and the reaction mixture was stirred at roomtemperature for about 16 hours. After removal of the solvent the residuewas redissolved in EtOAc and washed with water and brine. The organicphase was dried over MgSO₄ and the solvent evaporated. The residue waspurified using column chromatography (SiO₂, 9:1 n-hexanes/ethyl acetate)to give the desired product (2.11 g, 84% yield). ES-MS: 242 (M+1). Whenthe hydrochloride salt of an amine is used in place of thecyclopentylamine described above, 2 to 3 equivalents ofN,N-diisopropylethylamine and dichloromethane are used as solvent.

2.4-{[4-(Cyclopentylamino)-5-nitropyrimidin-2-yl]amino}trans-cyclohexan-1-ol

(2-Chloro-5-nitropyrimidin-4-yl)cyclopentylamine (6.18 mmol, 1.5 g) andtrans-4-aminocyclohexan-1-ol (7.42 mmol, 854 mg mL) were mixed in DMF(18 mL) and N,N-diisopropylethylamine (7.42 mmol, 1.29 mL) was added.The reaction mixture was stirred overnight. Solvent was removed in vacuoand the residue purified using column chromatography (SiO₂, 1:1n-hexanes/ethyl acetate→7:3 n-hexanes/ethyl acetate→ethyl acetate) togive the desired product (1.75 g, 88% yield). ES-MS: 322 (M+1). When thehydrochloride salt of an amine is used in place of thetrans-4-aminocyclohexan-1-ol described above, 2 to 3 equivalents ofN,N-diisopropylethylamine or sodium bicarbonate and tetrahydrofuran oracetonitrile were used as solvent.

3.4-{[5-Amino-4-(cyclopentylamino)pyrimidin-2-yl]amino}trans-cyclohexan-1-ol

4-{[4-(Cyclopentylamino)-5-nitropyrimidin-2-yl]amino}trans-cyclohexan-1-ol(2.18 mmol, 700 mg) was dissolved in 20 ml EtOH and hydrogenatedovernight at 1 bar with Pd/C (10%) as catalyst. The catalyst wasfiltered and the solvent evaporated to give the desired product (635 mg,100% yield) which was carried on to the next step without furtherpurification. ES-MS: 292 (M+1). This reduction can be also accomplishedusing the following procedure: Na₂S₂O₄ (140.0 mmol, 14 eq.) is dissolvedin 150 mL water and 75 mL dioxane and 7.5 mL NH₄OH solution are added.The corresponding nitro compound (10.0 mmol, 1 eq.) is added and thereaction mixture is stirred for 12 to 72 hours. Dioxane is evaporatedand the product is extracted by using EtOAc or brine/THF. The organicphase is dried over MgSO₄ and evaporated to give the desired product.

4.4-({8-[(2,6-Difluorophenyl)amino]-9-cyclopentylpurin-2-yl}amino)trans-cyclohexan-1-ol

4-{[5-Amino-4-(cyclopentylamino)pyrimidin-2-yl]amino}trans-cyclohexan-1-ol(1.13 mmol, 330 mg) was dissolved in DMF (8.5 mL) and2,6-difluorophenyl-isothiocyanate (1.13 mmol, 0.146 mL) was added. Thereaction mixture was stirred at room temperature for about 90 minutes.Ethanol (2.5 mL) was added and the reaction mixture was stirred forabout an additional 30 minutes. N,N-Diisopropylcarbodiimide (3.40 mmol,0.532 mL) was added and the reaction mixture was stirred overnight.Solvent was removed and the residue was purified using columnchromatography (SiO₂, 1:1 n-hexanes/ethyl acetate→ethyl acetate→1%methanol/ethyl acetate) to give the desired product (222.5 mg, 46%yield). ES-MS: 429 (M+1). Tetrahydrofuran can also be used as solvent inthis step.

Example 5.2 Synthesis oftrans-(4-Aminocyclohexyl){8-[2,4-difluorophenyl)amino]-9-cyclopentylpurin-2-yl}amine

1.trans-(4-Aminocyclohexyl){8-[2,4-difluorophenyl)amino]-9-cyclopentylpurin-2-yl}amine

N-[4-({8-[(2,4-difluorophenyl)amino]-9-cyclopentylpurin-2-yl}amino)trans-cyclohexyl](tert-butoxy)carboxamide(0.71 mmol, 375 mg) was dissolved in ethanol (6 mL) and cooled to 0° C.Acetyl chloride (3 mL) was added dropwise and the reaction was allowedto reach room temperature and stirred overnight. The precipitate wasfiltered off, washed with ethyl ether and dried under high vacuum toyield 372 mg (98% yield) as a trihydrochloride salt. ES-MS: 428 (M+1).

Alternatively,N-[4-({8-[(2,4-difluorophenyl)amino]-9-cyclopentylpurin-2-yl}amino)trans-cyclohexyl](tert-butoxy)carboxamidecan be dissolved in 9 mL of methylene chloride followed by the additionof 2.25 mL of TFA. The reaction mixture is stirred for about 2 hours.Solvent is removed in vacuo, the residue is redissolved in methylenechloride and neutralized with ammonium hydroxide. The solution is thenwashed with a saturated solution of sodium carbonate. The organic layeris separated and the aqueous layer is further extracted with methylenechloride. Combined organic layers are dried over sodium sulfate,filtered and the solvent removed in vacuo to yield the amine.

Example 5.3 Synthesis of8-(2-Fluorophenylamino)-2-(4-methoxyphenylamino)-9-(trans-4-(methylamino)cyclohexyl)-9H-purine

Boc-protected amine (481 mg, 0.88 mmol) was dissolved in THF (6 mL) andlithium aluminum hydride (1.0 M solution in THF, 2.64 mL, 2.64 mmol)added. The reaction mixture was heated at about 65° C. overnight. Thereaction mixture was cooled to 0° C. and quenched dropwise with wateruntil no further evolution of hydrogen was observed. The precipitate wasfiltered off and washed extensively with ethyl acetate. The solvent wasremoved in vacuo and the residue was purified using semi-preparativeHPLC (20% acetonitrile/water (0.1% TFA)→80% acetonitrile/water (0.1%TFA) over 30 min) to yield 191 mg of product.

Example 5.4 Synthesis of9-(trans-4-(Dimethylamino)cyclohexyl)-8-(2-fluorophenyl)-2-(4-methoxyphenyl)-9H-purine

Amine (200 mg, 0.359 mmol) was dissolved in a 1:1 mixture THF/methylenechloride (4 mL) and a solution of formaldehyde (37% in water, 53 μL,0.718 mmol) in THF (1 mL) was added dropwise, followed by sodiumtriacetoxyborohydride (761 mg, 3.59 mmol). The reaction mixture wasstirred at room temperature for 1 hour. The solvent was removed in vacuoand the residue was dissolved in DMSO/methanol (1:1 mixture) andpurified by semipreparative HPLC (20→70% acetonitrile/water (0.1% TFA)over 30 min). Fractions containing product were quenched with ammoniumhydroxide. After standing overnight, a precipitate formed and it wasfiltered and dried under high vacuum, to yield 108 mg of thedimethylamino compound (63% yield).

Example 5.5 Synthesis of(4-{8-[(2-Fluorophenyl)amino]-2-[(4-methoxyphenyl)amino]purin-9-yl}trans-cyclohexyl)methan-1-ol

Ethyl4-{8-[(2-fluorophenyl)amino]-2-[(4-methoxyphenyl)amino]purin-9-yl}-trans-cyclohexanecarboxylate(0.28 g, 0.55 mmol) was dissolved in 9 mL of THF and cooled to 0° C.(under nitrogen atmosphere). 1.38 mL of 1.0M LiAlH₄ in THF was addeddropwise. The solution turned a dark orange as the LiAlH₄ was added. Thereaction mixture was stirred for about 5 h and quenched by the additionof 40 mL of water. The reaction was extracted three times with ethylacetate. Organics were combined and dried with magnesium sulfate,filtered and the solvent was removed in vacuo. The crude reactionmixture was then purified using reverse-phase preparative HPLC (20-80%acetonitrile/water (0.1% TFA) over 30 min) to obtain 0.126 g of thedesired product (50% yield) after neutralization of the TFA salt. ES-MS:463 (M+1).

Example 5.6 Synthesis oftrans-4-{8-[(2-Fluorophenyl)amino]-9-[cis-4-(1-hydroxy-isopropyl)cyclohexyl]purin-2-yl}amino)cyclohexan-1-ol

Ethylcis-4-{8-[(2-fluorophenyl)amino]-2-[trans-(4-hydroxycyclohexyl)amino]purin-9-yl}cyclohexanecarboxylate (0.200 g, 0.4 mmol) was dissolved in 4 mL of dry THF. Methylmagnesium bromide (0.6 mL, 3.0M solution in diethyl ether, 4.0equivalents) was added dropwise at room temperature. The reactionmixture turned bright yellow and was stirred at room temperature forabout 1 hour. The completion of the reaction was monitored by LC-MS. Anadditional 4 equivalents of methyl magnesium grignard solution wereadded and the reaction mixture was heated overnight at about 30° C.

The reaction mixture was then cooled to room temperature and wasquenched slowly with saturated aqueous ammonium chloride solution. Thecrude was extracted in ethyl acetate and the extracts were dried overNa₂SO₄. The product was purified using column chromatography on silicagel using 1-4% (ethanol/ammonium hydroxide: 8:1) in dichloromethane. Thecompound was isolated as a light pink solid (57 mg, 29% yield).

Example 5.7 Synthesis ofcis-4-[8-[(2,6-Difluorophenyl)amino]-2-trans-({4-[4-methylpiperazinyl)carbonyl]cyclohexyl)amino}purin-9-ylcyclohexanecarboxylicacid N-(4-methylpiperazinyl)amide

Diester (10.0 mmol, 1 eq.) was dissolved in 100 mL THF and LiOH (200.0mmol, 20 eq.) (as a 1M aqueous solution) was added. The reaction mixturewas heated at about 60° C. overnight. After cooling to room temperature,the pH was adjusted to 4 by adding 6N HCl. Brine was added and phaseswere separated. The aqueous phase was extracted with THF and thecombined organic phases were dried over MgSO₄. The solvent wasevaporated to give the desired product.

Diacid (10.0 mmol, 1 eq.), HOBT (20.0 mmol, 2 eq.) and EDCI (24.0 mmol,2.4 eq.) were mixed in 100 mL DMF and stirred for 15 minutes. Amine(24.0 mmol, 2.4 eq.) was added and the reaction mixture was stirredovernight. The solvent was evaporated and the residue was purified usingHPLC.

Example 5.8 Synthesis of4-({9-[cis-4-(aminomethyl)cyclohexyl]-8-{(2,6-difluorophenyl)amino]purin-2-yl}trans-amino)cyclohexan-1-ol

1. cis-4-[(tert-Butoxy)carbonylamino]cyclohexane carboxylic acid

cis-4-Aminocyclohexyl carboxylic acid (2.0 g, 13.96 mmol) was dissolvedin 40 mL of 1,4-dioxane. Two equivalents of di-tert-butyl-dicarbonate(6.094 g, 27.92 mmol) were added followed by 3 equivalents of sodiumbicarbonate (4.06 g, 41.88 mmol) dissolved in 40 mL of water. Thereaction mixture was stirred at room temperature for about 12 hours. Thecompletion of the reaction was monitored by LC-MS. Saturated aqueousKHSO₄ was added dropwise, until gas evolution stopped. The solvent wasthen removed under reduced pressure and the crude product was extractedin ethyl acetate. The combined organic extracts were washed with aqueoussaturated KHSO₄ and dried over Na₂SO₄. The solvent was removed underreduced pressure, yielding 2.6 g of product. Based on ¹H NMR, theproduct was pure and used in subsequent steps without furtherpurification. ES-MS (m/z) 244.

2. cis-(tert-Butoxy)-N-[4-(hydroxymethyl)cyclohexyl]carboxamide

cis-4-[(tert-Butoxy)carbonylamino]cyclohexane carboxylic acid (2.6 g,10.68 mmol) was dissolved in THF (20 mL) and cooled to −10° C.(MeOH-ice). N-Methyl morpholine was added followed by isobutylchloroformate (1.175 mL, 10.68 mmol). After 10 min, NaBH₄ was added as asolid in one portion (1.213 g, 32.06 mmol). The reaction mixture waswarmed to 0° C. and methanol was added dropwise (13.35 mL). After 30min, the reaction was quenched with 5% aqueous KHSO₄. The reaction wasmonitored by LC-MS until complete. The crude product was extracted withethyl acetate and the combined extracts were dried over Na₂SO₄. Acolorless oil was obtained and solidified slowly at room temperature.The product and purity were assessed by LC-MS and ¹H NMR. No furtherpurification was necessary. (quantitative yield) ES-MS (m/z) 230.

3.cis-(tert-Butoxy)-N-{4-[(1,3-dioxobenzo[c]azolidin-2-yl)methyl]cyclohexyl}carboxamide

cis-(tert-Butoxy)-N-[4-(hydroxymethyl)cyclohexyl]carboxamide (0.5 g,2.18 mmol) and resin-bound triphenyl phosphine (1.453 g, 4.36 mmol, 3mmol/g resin) were suspended in 15 mL of dry THF. Phthalimide was addedin 5 mL of THF followed by diisopropyl azodicarboxylate (DIAD) (0.858mL, 4.36 mmol). The reaction was stirred at room temperature andmonitored by LC-MS. After overnight stirring at room temperature, theresin was removed by filtration and washed multiple times with 5 mLportions of THF. The filtrate combined with washings was concentratedunder reduced pressure. The product was purified using columnchromatography on silica gel using 10% ethyl acetate in hexanes aseluent. The product was isolated as a white solid (0.486 g, 1.35 mmol,62% yield) ES-MS (m/z) 359.

4. cis-2-[(4-Aminocyclohexyl)methyl]benzo[c]azolidine-1,3-dione

cis-(tert-Butoxy)-N-{4-[(1,3-dioxobenzo[c]azolidin-2-yl)methyl]cyclohexyl}carboxamide(0.486 g, 1.35 mmol) was suspended in ethanol (5 mL) and reacted withacetyl chloride (1 mL). The reaction mixture was stirred at roomtemperature for about 4 hours. The completion of the deprotection wasmonitored by LC-MS. The solvent was removed under reduced pressure andthe product was isolated as its HCl salt as a white solid and usedwithout further purification in the subsequent addition to2,4-dichloro-5-nitropyrimidine: ES-MS (m/z) 259.

5.4-({9-[cis-4-(Aminomethyl)cyclohexyl]-8-{(2,6-difluorophenyl)amino]purin-2-yl}trans-amino)cyclohexan-1-ol

2-[(4-{8-[(2,6-difluorophenyl)amino]-2-[trans-(4-hydroxycyclohexyl)-amino]purin-9-yl}cyclohexylmethyl]benzo[c]azolidine-1,3-dione(0.318 g, 0.52 mmol) was dissolved in ethanol (4.5 mL) and reacted withhydrazine (42 μL, 2.4 eq) at reflux temperature for about 5 hours. Awhite precipitate formed that was removed by filtration. The filtratecombined with washings of the precipitate, was concentrated underreduced pressure. The product was purified using column chromatographyon silica gel using 5-10% (ethanol/NH₄OH: 8/1) in dichloromethane as theeluent. The product was isolated as a white solid (198 mg, 80% yield).

Example 5.9 Synthesis of3-((trans-4-(8-(2,6-Difluorophenylamino)-9-cyclopentyl-9H-purin-2-ylamino)cyclohexyloxy)carbonyl)propanoicacid

trans-4-(8-(2,6-Difluorophenylamino)-9-cyclopentyl-9H-purin-2-ylamino)cyclohexanol(1 mmol, 1 eq.) and succinic anhydride (10 mmol, 10 eq.) were mixed in25 mL pyridine and stirred at room temperature for 3 days. The mixturewas heated at 50° C. for about 10 hours and the solvent was subsequentlyevaporated. The residue was recrystallized from acetone/MeOH to give thedesired product.

Example 5.10 Synthesis oftrans-4-(8-(2,6-Difluorophenylamino)-9-cyclopentyl-9H-purin-2-ylamino)cyclohexyl2-aminoacetate

trans-4-(8-(2,6-Difluorophenylamino)-9-cyclopentyl-9H-purin-2-ylamino)cyclohexanol(1 mmol, 1 eq.) DCC (2 mmol, 2 eq.), BOC-glycine (1.12 mmol, 1.12 eq.)and DMAP (1.12 mmol, 1.12 eq.) were mixed in 20 mL DCM and stirred atroom temperature for 2 days. Water and EtOAc were added, the phases wereseparated and the organic phase was dried over MgSO₄. Solvent wasevaporated and the residue was purified using column chromatography togive the desired Boc protected product.

The Boc protected product (1 mmol, 1 eq.) was dissolved in 15 mL DCM and4 ml TFA were added. The reaction mixture was stirred for about one hourand the solvent was evaporated. EtOAc and sat. NaHCO₃ solution wereadded and the phases separated. The organic phase was dried over MgSO₄and the solvent was evaporated. The residue was purified using columnchromatography/HPLC to give the desired product.

Example 5.11 Synthesis of3-(8-(2-Fluorophenylamino)-9-cyclopentyl-9H-purin-2-ylamino)benzamide

To a cooled solution (0° C.) of the cyano compound (100 mg, 0.24 mmol)in ethanol (1 mL), sodium hydroxide (18 mg, 0.46 mmol) and hydrogenperoxide (30%, 53 μL, 0.48 mmol) were added. The reaction mixture wasstirred for about 4 h at room temperature. Only starting material wasobserved by LCMS. Another 18 mg of sodium hydroxide and 53 μL ofhydrogen peroxide were added and the reaction mixture was stirred forabout another 8 h. Still only starting material was observed. Thereaction was heated to 60° C. for about 4 h. Product formation wasobserved together with traces of carboxylic acid. The reaction wasquenched to pH=7 with 6N HCl. The crude reaction mixture was purifiedusing semi-preparative reverse-phase HPLC (15% acetonitrile/water (0.1%TFA)→80% acetonitrile/water (0.1% TFA) over 30 min) to yield 35 mg ofamide as a solid after neutralization of the TFA salt. LRMS (ES) m/e 432[MH]⁺.

Example 5.12 Synthesis of2-((3-(2-(Piperidin-1-yl)ethoxy)phenyl)amino)-9-cyclopentyl-8-((2-fluorophenyl)amino)-9H-purine

In a round bottom flask, sodium hydroxide (0.585 g, 14.6 mmol) wasdissolved in 10 mL of water. THF (20 mL),3-{[4-(cyclopentylamino)-5-nitropyridin-2-yl]amino}phenol (1.15 g, 3.66mmol), and piperidyl ethyl chloride hydrochloride (0.81 g, 4.39 mmol)were added. The reaction mixture was heated at about 55° C. overnight.The reaction was monitored by LC-MS. The reaction mixture was poured inaqueous sodium bicarbonate solution and the crude was extracted in ethylacetate. The combined organic were dried over sodium sulfate andevaporated to dryness. The desired product was isolated as a solid(1.538 g, 98% yield) ES-MS (m/z) 427.3.

Example 5.13 Synthesis of8-((2-Fluorophenyl)amino)-2-((4-methoxyphenyl)amino)-9H-purine

The cyanoethyl substituted compound (0.17 mmol) was dissolved in amixture of THF:H₂O (8:2, 10 mL) and lithium hydroxide (1.05 mmol) wasadded. The reaction mixture was stirred at about 50° C. for about 72 h.The solvent was removed in vacuo and the residue was purified usingcolumn chromatography (SiO₂) or reverse-phase HPLC.

Example 5.14 Synthesis of4-({9-(2H-3,4,5,6-tetrahydropyran-4-yl)-8-[(2,4-difluorophenyl)amino]purin-2-yl}amino)thiane-1,1-dione

2H-3,4,5,6-Tetrahydropyran-4-yl[5-nitro-2-(thian-4-ylamino)pyrimidin-4-yl]amine

2H-3,4,5,6-Tetrahydropyran-4-yl(2-chloro-5-nitropyrimidin-4-yl)amine(3.14 mmol, 810.6 mg, obtained from 2,4-dichloro-5-nitropyrimidine and4-aminotetrahydropyran following the method described in Example 5.1)and 4-aminotetrahydrothiopyran (3.77 mmol, 441 mg, obtained followingthe procedure described in PCT Int. Appl. WO 2002083642) were dissolvedin DMF (20 mL). N,N-Diisopropylethylamine (3.77 mmol, 0.67 mL) was addedand the reaction was stirred at room temperature overnight. DMF wasremoved in vacuo and the crude was sonicated with ethyl acetate. Theprecipitate was filtered to yield the title compound (992 mg, 93%yield). ES-MS: 340 (M+1).

2H-3,4,5,6-tetrahydropyran-4-yl[5-amino-2-(thian-4-ylamino)pyrimidin-4-yl]amine

The title compound (760 mg, 93% yield) was obtained from2H-3,4,5,6-tetrahydropyran-4-yl[5-nitro-2-(thian-4-ylamino)pyrimidin-4-yl]amine(2.63 mmol, 892 mg) by catalytic hydrogenation following the proceduredescribed in Example 5.1, step 3. ES-MS: 310 (M+1).

[9-(2H-3,4,5,6-Tetrahydropyran-4-yl)-2-(thian-4-ylamino)purin-8-yl](2,4-difluorophenyl)amine

The title compound (577.1 mg, 71% yield) was obtained from2H-3,4,5,6-tetrahydropyran-4-yl[5-amino-2-(thian-4-ylamino)pyrimidin-4-yl]amine(1.81 mmol, 560 mg) and 2,4-difluorophenylisothiocyanate following theprocedure described in Example 5.1 step 4. ES-MS: 447 (M+1).

4-({9-(2H-3,4,5,6-tetrahydropyran-4-yl)-8-[(2,4-difluorophenyl)amino]purin-2-yl}amino)thiane-1,1-dione

[9-(2H-3,4,5,6-tetrahydropyran-4-yl)-2-(thian-4-ylamino)purin-8-yl](2,4-difluorophenyl)amine(1.2 mmol, 537 mg) was dissolved in methylene chloride (15 mL) and3-chloroperoxybenzoic acid (2.64 mmol, 591 mg) were added. The reactionwas stirred at room temperature for 18 h. The reaction mixture waswashed with saturated solution of sodium bicarbonate (10 mL) andextracted with chloroform (3×15 mL). The organic layer was dried overmagnesium sulfate and filtered. Solvent was removed in vacuo and theresidue was purified by column chromatography (SiO₂, 10% Methanol/ethylacetate) and reverse-phase HPLC (20% acetonitrile/water (0.1% TFA) to100% acetonitrile/water (0.1% TFA) over 30 min) to yield the titlecompound (146 mg, 25% yield). ES-MS: 479 (M+1).

Example 5.15 Synthesis of4-{8-[(2,4-difluorophenyl)amino]-2-[(4-trans-hydroxycyclohexyl)amino]purin-9-yl}thiane-1,1-dione

4-({8-[(2,4-Difluorophenyl)amino]-9-thian-4-ylpurin-2-yl}amino)-trans-cyclohexan-1-ol(0.49 mmol, 225 mg), obtained from 4-aminotetrahydrothiopyran (PCT Int.Appl. WO 2002083642), trans-4-aminocyclohexanol and 2,4-difluorophenylisothiocyanate following the general procedure described in Example 5.1,were dissolved in methylene chloride (5 mL), and 3-chloroperoxybenzoicacid (1.08 mmol, 241 mg) was added. The reaction was stirred at roomtemperature for 18 h. The reaction mixture was washed with saturatedsolution of sodium bicarbonate (5 mL) and extracted with chloroform(3×10 mL). The organic layer was dried over magnesium sulfate andfiltered. Solvent was removed in vacuo and the residue was purified bycolumn chromatography (SiO₂, ethyl acetate to 2% methanol/ethyl acetate)and reverse-phase HPLC (20% acetonitrile/water (0.1% TFA) to 100%acetonitrile/water (0.1% TFA) over 30 min) to yield the title compound(88.4 mg, 36% yield). ES-MS: 493 (M+1).

Example 5.16 Building Block Involved in the Synthesis of

(5S)-5-aminopiperidin-2-one, hydrochloride

(2S)-2-[(tert-Butoxy)carbonylamino]-4-(methoxycarbonyl)butanoic acid

L-Glutamic acid 5-methyl ester (91.3 mmol, 14.7 g) was added to asolution of triethylamine (274 mmol, 38 mL) in DMF (350 mL). Di-t-butyldicarbonate (183 mmol, 40 g) was added and the reaction was stirred at50° C. for 1 hour and then at room temperature overnight. Solvent wasremoved in vacuo and the crude material was purified by columnchromatography (SiO₂, 1:1 n-hexanes/ethyl acetate to ethyl acetate) toyield the title compound (20.36 g, 85% yield). ES-MS: 262 (M+1).

Methyl (4S)-4-[(tert-butoxy)carbonylamino]-5-hydroxypentanoate

In a round-bottom flask,(2S)-2-[(tert-butoxy)carbonylamino]-4-(methoxycarbonyl)butanoic acid (78mmol, 20.36 g) was dissolved in THF (300 mL). The solution was cooled to−10° C. and N-methylmorpholine (78 mmol, 8.58 mL) and ethylchloroformate (78 mmol, 7.48 mL) were added, followed by sodiumborohydride (234 mmol, 8.85 g). The reaction was stirred for 30 min atthis temperature and then quenched by slow addition of saturatedsolution of ammonium chloride until no further evolution of hydrogen wasobserved. The reaction mixture was then extracted with ethyl acetate anddried over magnesium sulfate. After filtration, solvent was evaporatedand the crude material was purified by column chromatography (SiO₂, 1:1n-hexanes/ethyl acetate) to yield the title compound (11.68 g, 61%yield). ES-MS: 248 (M+1).

Methyl (4S)-4-[(tert-butoxy)carbonylamino]-5-[(4-methylphenyl)sulfonyloxy]pentanoate

Methyl (4S)-4-[(tert-butoxy)carbonylamino]-5-hydroxypentanoate (9.11mmol, 2.25 g) was dissolved in 30 mL of methylene chloride.p-Toluenesulfonyl chloride (9.1 mmol, 1.7 g) and triethylamine (27.33mmol, 3.8 mL) were added and the reaction was stirred at roomtemperature overnight. Solvent was removed in vacuo and crude waspurified by column chromatography (SiO₂, 4:1 n-hexanes/ethyl acetate to7:3 n-hexanes/ethyl acetate) to yield the title compound (1.98 g, 54%yield). ES-MS: 402 (M+1).

Methyl (4S)-5-azido-4-[(tert-butoxy)carbonylamino]pentanoate

Methyl(4S)-4-[(tert-butoxy)carbonylamino]-5-[(4-methylphenyl)sulfonyloxy]pentanoate(4.93 mmol, 1.98 g) was dissolved in DMF (15 mL) and sodium azide (14.8mmol, 0.961 g) were added. The reaction was heated at 50° C. for 3hours. The reaction mixture was filtered and the solvent was removed invacuo. The crude was purified by flash chromatography (SiO₂, ethylacetate) to yield the title compound (1.07 g, 80% yield). ES-MS: 273(M+1).

N-((3S)-6-oxo(3-piperidyl))(tert-butoxy)carboxamide

Methyl (4S)-5-azido-4-[(tert-butoxy)carbonylamino]pentanoate (3.9 mmol,1.07 g) was dissolved in methanol (10 mL), and 10% palladium on carbon(0.1 g) was added. The reaction was stirred overnight under 1 atm ofhydrogen. The reaction was filtered and the solvent was removed in vacuoto yield the title compound (0.83 g, 99% yield). ES-MS: 215 (M+1).

(5S)-5-aminopiperidin-2-one, hydrochloride

N-((3S)-6-Oxo(3-piperidyl))(tert-butoxy)carboxamide (3.9 mmol, 0.83 g)were dissolved in ethanol (10 mL) and cooled to 0° C. Acetyl chloride (2mL) was added and the reaction was allowed to reach room temperature.The reaction was stirred for 1 hour after which the solvent was removedin vacuo to yield the title compound (725 mg, 99% yield) as thedihydrochloride salt. ES-MS: 115 (M+1).

Example 5.17 Building Block Used for the Synthesis of

(5R)-5-aminopiperidin-2-one, hydrochloride

The title compound was prepared as described in Example 5.15, startingfrom D-glutamic acid 5-methyl ester.

Example 5.18 Building Block Used for the Synthesis of

Synthesis of 6-chloro-2-fluorobenzeneisothiocyanate

A solution of 2-chloro-6-fluoroaniline (767 mg, 5.29 mmol) intetrahydrofuran (5 ml) was added drop wise with stirring to a solutionof di-2-pyridyl thionocarbonate (2.46 g, 10.58 mmol) in tetrahydrofuran(7 ml) at room temperature. The reaction mixture was stirred for 60hours at room temperature and then the solvent was evaporated. Theresulting residue was purified by chromatography on a normal phasesilica gel column with pentane. Fractions containing clean product werecombined and the solvent evaporated to give the title compound (167 mg,17%): ¹H NMR (400 MHz, CDCl₃) δ 7.19-7.23 (m, 1H), 7.12-7.19 (m, 1H),7.04-7.10 (m, 1H).

Example 5.19 Building Block Used for the Synthesis of

Synthesis of 3-fluoropyridin-2-isothiocyanate

A solution of 3-fluoro-pyridin-2-ylamine (928 mg, 8.28 mmol) indichloromethane (3 mL) was added dropwise with stirring to a solution ofthiophosgene (1.9 mL, 24.83 mmol) in dichloromethane (6 mL) at 0° C. Thereaction mixture was stirred for 1 hour at room temperature. Thereaction was diluted with dichloromethane and saturated aqueous sodiumbicarbonate. The organic layer was separated and the aqueous solutionextracted 3 times with dichloromethane. The organic layers were combinedand the solvent evaporated. The resulting residue was purified bychromatography on a normal phase silica gel column with 10% ethylacetate in hexanes. Fractions containing clean product were combined andthe solvent evaporated to give the title compound (479 mg, 37%): ¹H NMR(400 MHz, CDCl₃) δ 8.22-8.23 (m, 1H), 7.48-7.52 (m, 1H), 7.21-7.26 (m,1H).

Example 5.20 Building Block Used for the Synthesis of

Synthesis of methyl (2E)(4S)-4-aminopent-2-enoate hydrochloride

(2S)-2-[(tert-Butoxy)carbonylamino]-N-methoxy-N-methylpropanamide

To a solution of Boc-alanine (20 grams, 105.7 mmol) in dichloromethane(170 ml) was added HOBT (14.28 g, 105.7 mmol) andN,O-dimethylhydroxylamine hydrochloride (10.31 g, 105.7 mmol). Themixture was chilled with an ice water bath then triethylamine (30 ml,211.4 mmol) and 1,3-dicyclohexylcarbodiimide (21.81 g, 105.7 mmol) wereadded. The reaction was stirred in the ice water bath for 1 hour andthen allowed to warm to room temperature overnight. The crude reactionwas then chilled in an ice water bath and the precipitate filtered. Theresulting organic solution was then washed twice with 1N aqueous sodiumhydroxide (50 mL), twice with 10% aqueous citric acid (50 mL), and oncewith brine. The solution was then dried over anhydrous sodium sulfate,filtered, and the solvent evaporated. The resulting residue was purifiedby chromatography on a normal phase silica gel column with 30-100% ethylacetate in hexanes. Fractions containing clean product were combined andthe solvent evaporated to give the title compound (20 g, 81%): ES-MS(m/z) 233.2 [M+1]⁺.

Methyl (2E)(4S)-4-[(tert-butoxy)carbonylamino]pent-2-enoate

A solution of(2S)-2-[(tert-butoxy)carbonylamino]-N-methoxy-N-methylpropanamide (13.05g, 56.18 mmol) in ethyl ether (560 mL) was chilled with an ice waterbath and then 95% lithium aluminum hydride (2.80 g, 70.23 mmol) wasadded. The reaction was stirred at room temperature for 20 minutes andthen a solution of aqueous potassium hydrogen sulfate (300 mL, 0.33M)was added. The resulting mixture was extracted three times with ethylether. The combined organic layers were washed three times with 1Nhydrogen chloride, three times with saturated aqueous sodium hydrogencarbonate, and once with brine. The solution was then dried overanhydrous sodium sulfate, filtered, and the solvent evaporated. Theresulting solid was dissolved in anhydrous tetrahydrofuran (430 mL) thenadded to a cold solution of trimethyl phosphonoacetate (27.3 mL, 168.5mmol) and sodium hydride (112 mmol) in anhydrous tetrahydrofuran (130mL) that had been previously stirred at room temperature for 30 minutes.The reaction was stirred for 5 minutes in a ice water bath, at roomtemperature for 20 minutes, and then water (500 mL) was added. Thereaction mixture was diluted with brine and ethyl acetate, stirred, andthe layers separated. The organic layer was dried over anhydrous sodiumsulfate, filtered, and the solvent evaporated. The resulting residue waspurified by chromatography on a normal phase silica gel column with0-30% ethyl acetate in hexanes. Fractions containing clean product werecombined and the solvent evaporated to give the title compound (8.85 g,69%): ES-MS (m/z) 230.4 [M+1]⁺.

Methyl (2E)(4S)-4-aminopent-2-enoate hydrochloride

A solution of methyl(2E)(4S)-4-[(tert-butoxy)carbonylamino]pent-2-enoate (3.083 g, 13.45mmol) in 4N hydrogen chloride in dioxane was stirred at room temperaturefor 1 hour. The volatiles were evaporated to give the title compound(2.2 g, 98%): ES-MS (m/z) 130.3 [M+1]⁺.

Methyl(4S)-4-({5-amino-2-[(methylethyl)amino]pyrimidin-4-yl}amino)pentanoate

A solution of methyl (2E)(4S)-4-aminopent-2-enoate hydrochloride (1.7 g,10.31 mmol) in tetrahydrofuran (7 mL) was added drop wise to a solutionof 2,4-dichloro-5-nitropyrimidine (2.0 g, 10.31 mmol) anddiisopropylethylamine (3.6 mL, 20.6 mmol) in tetrahydrofuran (17 mL)chilled at −78° C. The reaction was stirred at −78° C. for 1 hour andthen at room temperature overnight. The was solvent was evaporated andthe resulting residue was purified by chromatography on a normal phasesilica gel column with 0-20% ethyl acetate in hexanes. Fractionscontaining clean product were combined and the solvent evaporated togive 2.35 g white solid. To the solid were added anhydrousN,N-dimethylformamide (40 mL), diisopropylethylamine (1.44 mL, 8.25mmol), and isopropylamine (0.70 mL, 8.25 mmol). The mixture was stirredat room temperature for 70 hours, diluted with water, and extractedthree times with dichloromethane. The organic layers were combined,dried over anhydrous sodium sulfate, filtered, and the solventevaporated. To the resulting oil was added anhydrous ethanol (50 mL) and10% palladium on carbon (200 mg). The solution was treated with hydrogengas from a balloon and stirred at room temperature overnight. Thereaction mixture was filtered and the solvent evaporated to provide thetitle compound (2.24 g, 77%): ES-MS (m/z) 282 [M+1]⁺.

(4S)-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)aminopurin-9-yl}pentanamide

A solution of methyl(4S)-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}pentanoate(500 mg, 1.15 mmol) in anhydrous methanol (25 mL) at −78° C. wassaturated with ammonia gas. The solution was sealed in a reaction tubeand allowed to warm to room temperature followed by heating at 40° C.for 2 days. The solvent was evaporated and the resulting residue waspurified by chromatography on a normal phase silica gel column with70-100% ethyl acetate in hexanes. Fractions containing clean productwere combined and the solvent evaporated to give the title compound (223mg, 46%): ES-MS (m/z) 422.3 [M+1]⁺.

Example 5.21 Building Block Used for the Synthesis of

Synthesis of methyl (2E)(4R)-4-aminopent-2-enoate hydrochloride

(2R)-2-[(tert-Butoxy)carbonylamino]-N-methoxy-N-methylpropanamide

The title compound was prepared as(2S)-2-[(tert-butoxy)carbonylamino]-N-methoxy-N-methylpropanamide withboc-D-alanine (20 grams, 105.7 mmol) to give the title compound (21.7 g,88%): ES-MS (m/z) 233.2 [M+1]⁺.

Methyl (2E)(4R)-4-[(tert-butoxy)carbonylamino]pent-2-enoate

The title compound was prepared as Methyl(2E)(4S)-4-[(tert-butoxy)carbonylamino]pent-2-enoate with(2R)-2-[(tert-butoxy)carbonylamino]-N-methoxy-N-methylpropanamide (13.05grams, 56.18 mmol) to give the title compound (10.2 g, 79%): ES-MS (m/z)230 [M+1]⁺.

Methyl (2E)(4i)-4-aminopent-2-enoate hydrochloride

A solution of methyl(2E)(4R)-4-[(tert-butoxy)carbonylamino]pent-2-enoate (3.61 g, 15.75mmol) in 4N hydrogen chloride in dioxane was stirred at room temperaturefor 1 hour. The volatiles were evaporated to give the title compound(2.6 g, 98%): ES-MS (m/z) 130.3 [M+1]⁺.

Methyl(4R)-4-({5-amino-2-[(methylethyl)amino]pyrimidin-4-yl}amino)pentanoate

The title compound was prepared as methyl(4S)-4-({5-amino-2-[(methylethyl)amino]pyrimidin-4-yl}amino)pentanoatewith methyl (2E)(4R)-4-aminopent-2-enoate hydrochloride (1.7 g, 10.31mmol) to give the title compound (2.17 g, 75%): ES-MS (m/z) 282 [M+1]⁺.

(4R)-4-{2-[(Methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}pentanamide

The title compound was prepared as(4S)-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}pentanamidewith methyl(4R)-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}pentanoate(500 mg, 1.15 mmol) to give the title compound (273 mg, 57%): ES-MS(m/z) 422.3 [M+1]⁺.

Example 5.22 Building Block Used for the Synthesis of

Synthesis of 4-Aminopiperidyl pyrrolidinyl ketone

(tert-Butoxy)-N-[1-(pyrrolidinylcarbonyl)(4-piperidyl)]carboxamide

1-Pyrrolidine carbonylchloride (1.10 g, 9.99 mmol) was dissolved in 400ml if dichloromethane under N₂. (Tert-butoxy)-N-(4-piperidyl)carboxamide(2.0 g, 9.99 mmol) and triethyl amine (1.40 mL, 9.99 mmol) were addedand the reaction mixture was stirred for three days. The reaction wasquenched with sat. NaHCO₃ solution and extracted with dichloromethane.The combined organic phases were dried over MgSO₄ and the solventevaporated to give the product as a white solid. (2.63 g, 8.84 mmol,89%).

4-Aminopiperidyl pyrrolidinyl ketone

(tert-Butoxy)-N-[1-(pyrrolidinylcarbonyl)(4-piperidyl)]carboxamide (2.0g, 6.73 mmol) was dissolved in 40 mL dichloromethane and trifluoroaceticacid (15 mL, 201.94 mmol) were added. The reaction mixture was stirredfor 4 hours. The solvent was evaporated to give the product as a lightbrown semi-solid, which was used directly for the next step. (2.09 g,6.73 mmol, 100%).

Example 5.23 Synthesis of4-[(R)-8-(2,4-difluoro-phenylamino)-9-(4-hydroxy-cyclohexyl)-7H-purin-2-ylamino]-cyclohexanone

4-[(R)-8-(2,4-Difluoro-phenylamino)-2-(1,4-dioxa-spiro[4.5]dec-8-ylamino)-7H-purin-9-yl]-cyclohexanol(0.62 g, 1.7 mmol) was dissolved in 25 mL of methylene chloride under anN2 atmosphere. Trifluoroacetic acid (5 mL) was then added dropwise viaaddition funnel. After stirring for 24 h the resulting reaction mixturewas concentrated under reduced pressure. Saturated sodium bicarbonatewas added to the resulting residue until pH 12. The basic aqueous layerwas then extracted using chloroform (2×75 mL). The combined organiclayers were dried with MgSO4. The crude product was then purified on thepreparatory HPLC using a 5-70% CH3CN/H2O over 39 minutes method.Fractions of greater then 98% purity via analytical HPLC were combinedand concentrated. Excess trifluoroacetic acid was removed by washing theproduct with 1.75 M potassium carbonate (3×100 mL). The organic layerswere then concentrated to dryness under vacuum to give the ketone (0.015g 0.033 mmol, 12%) as a fine white powder: LC-MS (m/z) 457.1 [M+1]⁺.

Example 5.24 Building Block Used for the Synthesis of

Synthesis of (S)-(−)-4-amino-2-pyrrolidinone

(S)-(−)-4-azido-2-pyrrolidinone

To an ice cooled solution of (R)-(+)-4-hydroxy-2-pyrrolidinone (25.0 g,247 mmol) in dichloromethane (300 mL) was added triethylamine (17.0 g,168.7 mmol) and methane sulfonyl chloride (21 mL, 272 mmol) dropwise.The solution was stirred at ambient temperature for one hour. Thereaction was monitored via TLC (100% ethyl acetate using permanganatestain). The solution was then condensed under reduced pressure to give asolid. The solid was diluted with DMF (300 mL) followed by the additionof sodium azide (48.24 g, 742 mmol). The solution was heated to 60° C.for 3 hours. The reaction was monitored via TLC (100% ethyl acetateusing permanganate stain). The solution was then condensed under reducedpressure and the resultant oil purified via silica gel chromatography(50-80% acetate/hexanes followed by 12% methanol/dichloromethane) toafford the title compound (10.2 g, 32%). ¹H-NMR (CD₃OD) δ 4.43 (m, 1H),3.71 (dd, 1H), 3.34 (m, 1H), 2.75 (dd, 1H), 2.29 (dd, 1H).

(S)-(−)-4-amino-2-pyrrolidinone

To a solution of (S)-(−)-4-azido-2-pyrrolidinone (10.2 g, 80.8 mmol) inTHF (450 mL) was added triphenylphosphine resin bound (40.5 g, 3 mmolcomp/1.0 g resin). The solution was heated to 60° C. for two hours. Theevolution of nitrogen gas from the solution is an indicator of thereaction proceeding. The reaction is monitored via TLC and permanganatestain for completion. The solution was filtered through a glass frit andthe resin bound product is then added to another reaction vessel anddiluted with water (500 mL). The solution was heated to 70° C. forsixteen hours. The solution filtered through a glass frit and theaqueous filtrate was condensed under reduced pressure and chased withtoluene (3×) to afford the title compound upon vacuum (5.62 g, 62%).¹H-NMR (CD₃OD) δ 3.68 (m, 1H), 3.56 (m, 1H), 3.04 (m, 1H), 2.54 (m, 1H),2.05 (m, 1H).

Example 5.25 Synthesis of5-[9-Cyclopentyl-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino]pyridine-2-ol

9-Cyclopentyl-N2-(6-methoxypyridin-3-yl)-N8-(2,4,6-trifluorophenyl)-9H-purine-2,8-diamine(0.350 g, 0.769 mmol) was dissolved in 30% HBr/acetic acid in a sealedtube and heated to 80 oC for 16 hours. Product confirmed by LC-MS. Thesolution was partitioned between 1.75 M potassium carbonate and ethylacetate (3×). The organics were combined, dried over magnesium sulfate,filtered and solvent removed under reduced pressure. The resultant solidwas purified via preparative HPLC (5-55% acetonitrile/water, 20 mL/min.)to afford the title compound (0.212 g, 34%). ES-MS (m/z) 442 [M+1]+.Melting point 257-260° C.

Example 5.26 Building block used for the synthesis of(S)-3-[8-(2,4-trifluoro-phenylamino)-2-isopropylamino-purin-9-yl]-butyramide

(S)-(−)-3-Aminobutyramide hydrochloride

To a solution of (3S)-3-[tert-butoxycarbonyl)amino]butanoic acid (2 g,9.8 mmol) in acetonitrile (10 mL) was added HBTU (4.8 g, 12.7 mmol),ammonium chloride (2.6 g, 49 mmole) at room temperature. The reactionmixture was cooled to 0° C. and diisopropylethyl amine (10.0 g, 78mmole). The ice-water bath was removed and the brown mixture was stirredunder nitrogen for 12 hours. The solvent was removed in vacuo and theresidue was dissolved in dichloromethane (100 mL). The organic phase waswashed with sodium carbonate aqueous solution (saturated). The organicphase was dried with brine followed by sodium sulfate, which wassubsequently filtered. The organic phase was concentrated and purifiedby normal phase silica gel chromatography (50% ethyl acetate/hexanefollowed by 10% methanol/dichloromethane) to afford partially purifiedfractions, which were combined and used in the next reaction. The crudeamide was dissolved in 10 mL dry dioxane and cooled to 0° C. in anice/water bath. 4N HCl in dioxane solution (12.2 mL, Aldrich) was addeddropwise and the mixture was stirred for 3 hours at room temperature.The solvent was removed in-vacuo to afford oily solid which was notpurified further but was suspended in THF (5 mL). Diisopropylethyl amine(2.53 g, 19.6 mmole) was added to create a slurry.

(S)-3-(2-Chloro-5-nitro-pyrimidin-4-ylamino)-butyramide

2,4-dichloro-5-nitropyrimidine (1.9 g, 9.8 mmole) was added to aoven-dried 100 ml round-bottomed flask and THF (27 mL) was added toafford a solution. The mixture was cooled to −78° C. under nitrogenatmosphere and the slurry (Step A) was added dropwise. The reactionmixture was stirred at −78° C. for 30 minutes and then warmed to roomtemperature over 3 h. Water (10 mL) was added to the mixture and theorganic solvent was removed in vacuo. The aqueous phase was extractedwith ethyl acetate (3×50 mL) and the resulting organic phase was driedwith brine. The organic phase was concentrated to a residue. Normalphase silica gel chromatography (5-50% ethyl acetate/hexane) of theresidue afforded the title compound (761 mg, 30% overall): ES-MS (m/z/)260.0 [M+1]⁺. The intermediate was employed according to the standardprocedure to provide(S)-3-[8-(2,4,6-trifluoro-phenylamino)-2-isopropylamino-purin-9-yl]-butyramide.

Example 5.27 Building block used for the synthesis of(R)-3-[8-(2,4,6-trifluoro-phenylamino)-2-isopropylamino-purin-9-yl]-butyramide

(R)-3-(2-Chloro-5-nitro-pyrimidin-4-ylamino)-butyramide was similarlyprepared (586 mg, 23% overall yield): ES-MS (m/z/) 260.0 [M+1]⁺. Theintermediate was employed according to the standard procedure to provide(R)-3-[8-(2,4,6-trifluoro-phenylamino)-2-isopropylamino-purin-9-yl]-butyramide.

Example 5.28 Building block used for the synthesis of4-[9-(R)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-yl)-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]-cyclohexanol

Preparation of (R)-tetrahydro-3-thiopheneamine hydrochloride

Synthesis of title compound was performed according to Dehmlow, E. V etal. Synthesis 1992, 10, 947-9. Amine hydrochloride was employed in usualmanner to afford4-[(R)-9-tetrahydro-thiophen-3-yl-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]cyclohexanol.

Synthesis of4-[9-(R)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-yl)-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]-cyclohexanol

4-[(R)-9-Tetrahydro-thiophen-3-yl-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]cyclohexanol(100 mg, 0.21 mmole) was dissolved in MeOH (1 mL) and the mixture wascooled to 0° C. with ice/water bath. Oxone (338 mg, 0.52 mmole) wasdissolved in water (1 mL) and the solution was added dropwise to theformer mixture at 0° C. with vigorous stirring. The bath was thenremoved and the cloudy mixture was stirred at room temperature for 10minutes. The mixture was added to dichloromethane (100 mL) and theorganic phase was washed with sodium carbonate (aqueous), brine anddried over sodium sulfate. After filtration, the solvent was removed andthe residue was subjected to silica gel chromatography (5-10% methylenechloride/methanol) to afford sulfone (59 mg, 57%): ES-MS (m/z) 497.0[M+1]⁺.

Example 5.29 Building block used for the synthesis of4-[9-(S)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-yl)-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]-cyclohexanol

Preparation (S)-tetrahydro-3-thiopheneamine hydrochloride

Synthesis of title compound was performed according to Dehmlow, E. V.;Westerheide, R.; Synthesis 1992, 10, 947-9. Amine hydrochloride wasemployed in usual manner to afford4-[(S-9-Tetrahydro-thiophen-3-yl-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]cyclohexanol.

Synthesis of4-[9-(S)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-yl)-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]-cyclohexanol

Synthesis of sulfone was similarly performed to afford4-[9-(S)-1,1-dioxo-tetrahydro-1λ⁶-thiophen-3-yl)-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]-cyclohexanol(51 mg, 49%): ES-MS (m/z/) 497.0 [M+1]⁺.

Example 5.30 Building Block Used for the Synthesis of

Synthesis of4-[9-((1S,2R)-2-methyl-cyclopentyl)-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]-cyclohexanol.Preparation of cyclopentanamine, 2-methyl-, hydrochloride, (1S,2R)-(9CI)

Synthesis of title compound was performed according to Wiehl, W.; Frahm,A. W.; Chemische Berichte 1986 119(8), 2668-77. Amine hydrochloride wasemployed in usual manner.

Example 5.31 Building block used for the synthesis of4-[9-((1R,2S)-2-Methyl-cyclopentyl)-8-(2,4,6-trifluoro-phenylamino)-9H-purin-2-ylamino]-cyclohexanol

Preparation of cyclopentanamine, 2-methyl-, hydrochloride, (1R,2S)-(9CI)

Synthesis of title compound was performed according to Wiehl, W.; Frahm,A. W.; Chemische Berichte 1986 119(8), 2668-77. Amine hydrochloride wasemployed in usual manner.

Example 5.32 Building Block Used for the Synthesis of

Synthesis of methyl 4-amino-1-hydroxycyclohexanecarboxylatehydrochloride

((1S)-1-Phenylethyl)(1,4-dioxaspiro[4.5]dec-8-yl)amine

1,4-Dioxaspiro[4.5]decan-8-one (10 g, 64.03 mmol) was dissolved in drydichloroethane (300 mL) under an atmosphere of nitrogen.(1S)-1-phenylethylamine (8.96 mL, 70.43 mmol) was added neat at roomtemperature followed by sodium triacetoxyborohydride (20.36 g, 96.04mmol) neat in small portions. The reaction was stirred at roomtemperature overnight. The reaction was quenched by the addition ofdistilled water (200 mL). The phases were separated and the aqueousphase was extracted with dichloromethane. The combined organic phaseswere dried over sodium sulfate. The filtrate was concentrated underreduced pressure. A yellow oil was obtained of satisfactory purity basedon (11.2 g, 67% yield). M+1: 262.

N-((1S)-1-Phenylethyl)-N-(1,4-dioxaspiro[4.5]dec-8-yl)-2,2,2-trifluoroacetamide

((1S)-1-Phenylethyl)(1,4-dioxaspiro[4.5]dec-8-yl)amine (11.2 g, 42.85mmol) was dissolved in dichloromethane (135 mL) at room temperature. Thesolution was treated with pyridine (3.81 mL, 47.14 mmol) andtrifluoroacetic anhydride (7.15 mL, 51.42 mmol). The reaction wasstirred over the week-end at room temperature. The completion of thereaction was ascertained by LC-MS. The reaction was washed withsaturated ammonium chloride. After drying over sodium sulfate, theorganic extracts were concentrated to a yellow oil. The crude was usedwithout further purification.

N-((1S)-1-Phenylethyl)-2,2,2-trifluoro-N-(4-oxocyclohexyl)acetamide

N-((1S)-1-Phenylethyl)-N-(1,4-dioxaspiro[4.5]dec-8-yl)-2,2,2-trifluoroacetamide(15.31 g, 42.84 mmol) was dissolved in tetrahydrofuran (30 mL). Thesolution was treated with 30 mL of 3.0 N aqueous HCl. The reaction washeated to 50-60° C. over 48 h. The reaction was cooled to roomtemperature. THF was removed under reduced pressure. The crude productwas extracted with dichloromethane and was purified by silica gel column(eluent 15-20% ethyl acetate in hexanes). The product was isolated as ayellow oil (5.49 g, 41% yield) M+1: 314.

N-((1S)-1-Phenylethyl)-N-[4-(1,1-dimethyl-1-silaethoxy)-4-cyanocyclohexyl]-2,2,2-trifluoroacetamide

N-((1S)-1-Phenylethyl)-2,2,2-trifluoro-N-(4-oxocyclohexyl)acetamide (3.8g, 12.13 mmol) was dissolved in 30 mL of dichloromethane. ZnI₂ (0.774 g,2.42 mmol) was added to the solution as a solid at room temperaturefollowed by trimethylsilyl chloride (3.25 mL, 24.25 mmol). The reactionmixture was heated to reflux temperature. The conversion was monitoredby LC-MS. After 4 h, heating was stopped and the solvent was removedunder reduced pressure. 50 mL of dry diethyl ether were added. Theresulting cloudy suspension was evaporated to dryness. The resultingorange oil was re-suspended in 100 mL of diethyl ether. A small amountof white solid was removed by filtration and was washed with a smallvolume of diethyl ether. The combined filtrates were evaporated todryness and the residue was maintained under high vacuum overnight. Theproduct was used without further purification (5.64 g). M+1: 413.

4-[N-((1S)-1-Phenylethyl)-2,2,2-trifluoroacetylamino]-1-hydroxycyclohexanecarboxamide

N-((1S)-1-Phenylethyl)-N-[4-(1,1-dimethyl-1-silaethoxy)-4-cyanocyclohexyl]-2,2,2-trifluoroacetamide(5.64 g, 13.67 mmol) was suspended in 15 mL of concentrated hydrochloricacid. The reaction was stirred at room temperature for 1.5 daysresulting in the formation of a dark orange suspension. The solid wascollected by filtration, dissolved in 10 mL of methanol under mildtemperature and slowly was precipitated out with water. (lightly coloredsolid separating from orange solution). The mother liquor was collectedconcentrated and the precipitation conditions were reproduced. Thisisolation step yielded overall 2.6 g of light yellow solid (53%) cleanby ¹H and ¹⁹F NMR. M+1: 359.

Methyl-cis-4-amino-1-hydroxycyclohexanecarboxylate hydrochloride

4-[N-((1S)-1-Phenylethyl)-2,2,2-trifluoroacetylamino]-1-hydroxycyclohexanecarboxamide(2.6 g, 7.25 mmol) was suspended in 30 mL of concentrated hydrochloricacid and the reaction mixture was heated to 80° C. for 6 h (light yellowsolution). The completion of the reaction was assessed by LC-MS. Thereaction mixture was cooled to room temperature and 40 mL of methanolwere added. The solution was stirred at room temperature for 36 h.Methanol was removed under reduced pressure. Organic side products wereremoved by extraction in diethyl ether. The aqueous solution wasconcentrated under reduced pressure and the residue was dried overnight.Methyl-cis-4-amino-1-hydroxycyclohexanecarboxylate hydrochloride wasisolated as a solid and was used without further purification.(quantitative yield).

Example 5.33 Synthesis of1-hydroxy-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}cyclohexanecarboxylicacid

Methyl1-hydroxy-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}cyclohexanecarboxylate(1.858 g, 3.858 mmol) was dissolved in 27 mL of 4.0 N aqueoushydrochloric acid solution. The reaction mixture was heated to 60° C.for 24 h. The mixture was then concentrated under reduced pressure to anoil and purified by preparative HPLC (20-80% acetonitrile-water, 0.1%TFA). The product was isolated as a white solid by filtration afterevaporating acetonitrile from the combined fractions and neutralizingwith concentrated ammonium hydroxide. (1.325 g, 73% yield).

Example 5.34 Building block used for the synthesis ofN-cyclopentyl(1-hydroxy-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}cyclohexyl)carboxamide

1-Hydroxy-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}cyclohexanecarboxylicacid (0.200 g, 0.4 mmol) was dissolved in 4 mL of dry THF. Cyclopentylamine (0.079 mL, 0.8 mmol) was added neat followed by di-isopropyl amine(0.105 mL, 0.6 mmol). Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP) was added last, as a solid in one portion atroom temperature (0.177 g, 0.4 mmol). The reaction was complete within10 min as confirmed by LC-MS. DMF was removed under reduced pressure.The residue was triturated in saturated aqueous sodium bicarbonate. Theresulting beige solid was collected by filtration and washed with water.The crude product was re-crystallized from hot methanol-water. Thecrystals were dried in vacuum oven. (141 mg, 66% yield) M+1: 532.

Example 5.35 Building block used for the synthesis of1-(hydroxymethyl)-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}cyclohexan-1-ol

Methyl1-hydroxy-4-{2-[(methylethyl)amino]-8-[(2,4,6-trifluorophenyl)amino]purin-9-yl}cyclohexanecarboxylate(0.300 g, 0.6 mmol) was dissolved in 3 mL of dry methanol. The solutionwas cooled to 0° C. before addition of solid sodium borohydride (0.300g, 7.92 mmol). After 1 h at low temperature, the reaction was warmed tort and stirred overnight. The reaction was quenched with 5 mL of asaturated solution of ammonium chloride. The crude product was extractedwith dichloromethane (four times). The product was purified by columnchromatography (75% ethyl acetate in hexanes) followed bysemi-preparative HPLC. The fractions were neutralized using aresin-exchange column. (0.101 g, 37% yield). M+1: 451.

Example 5.36 Building Block Used for the Synthesis of

Synthesis of (3R)-3-aminobutanol

tert-Butyl (3R)-3-{benzyl[(1R)-1-phenylethyl]amino}butanoate

n-BuLi (29.5 mL, 47.3 mmol) was added via canula to a solution of(R)—(N-benzyl)[N-(1-phenyl)ethyl]amine (10.0 g, 47.3 mmol) in THF (75mL) at 0° C. under N₂. The reaction was stirred for 20 minutes, andsubsequently cooled to −78° C. tert-Butyl crotonate (3.5 g, 24.6 mmol)dissolved in THF (30 mL) was added to the cooled reaction mixture over20 minutes. After 75 minutes, the reaction was quenched with saturatedaqueous NH₄Cl and brine was then added. The layers were separated andthe aqueous layer was further extracted with Et₂O. The organics werecombined, dried with MgSO₄, filtered, and concentrated to a yellow crudeoil. The crude product was dissolved in hexanes (100 mL) and washed with10% aqueous citric acid solution (3×25 mL). The organics were pooled,dried with MgSO₄, filtered and condensed to yield 6.2 g (17.55 mmol,37%) of the title compound.

(3R)-3-{benzyl[(1R)-1-phenylethyl]amino}butanol

tert-Butyl (3R)-3-{benzyl[(1R)-1-phenylethyl]amino}butanoate (6.2 g,17.6 mmol) was dissolved in THF (100 mL). The 1 L-flask was purged withN₂ and cooled to 0° C. Lithium aluminum hydride (2.7 g, 69.8 mmol) wasslowly added over 5 minutes. The reaction was allowed to stir at 0° C.for 1 hour, and then heated to 60° C. for 1 hour. The reaction wascooled to room temperature and diluted with Et₂O (500 mL). This solutionwas quenched with a mixture of celite:Na₂SO₄ 10H₂O (1:1) added over 15minutes. The solution was then filtered and the mother liquor condensedto yield 3.9 g (13.8 mmol, 78%) of the title compound.

(3R)-3-Aminobutanol

(3R)-3-{benzyl[(1R)-1-phenylethyl]amino}butanol (3.9 g, 13.8 mmol), wasdissolved in methanol (60 mL). Pearlman's catalyst was added to thereaction and subsequently pressurized to 30 psi with H₂ on a Parrshaker. After 24 hours, the reaction was filtered through celite andwashed additionally with methanol (150 mL). This mixture was condensedto yield 1.2 g (13.4 mmol) of the title product.

Example 5.37 Building Block Used for the Synthesis of

Synthesis of trans-4-amino-1-methylcyclohexanol

trans-4-Dibenzylaminocyclohexanol

To a solution of trans-4-aminocyclohexanol (7.90 g, 68.5 mmol) inacetonitrile (150 mL), was added cesium carbonate (51.4 g, 157.5 mmol)and benzyl bromide (18.2 g, 143.8 mmol). The solution was stirred atambient temperature for 16 hours. The solution was complete by LC-MS andthe mixture filtered through a frit, washed with additionalacetonitrile, and condensed under reduced pressure. The solid waspartitioned between water and dichloromethane (500 mL) and dried oversodium sulfate, filtered and solvent removed under reduced pressure toafford the title compound (17.14 g, 85%). ES-MS (m/z) 296.5 [M+1]⁺.

trans-4-Dibenzylaminocyclohexanone

Oxalyl chloride (12.89 g, 101.1 mmol) in dichloromethane (200 mL) wascooled to −78° C. DMSO (14.5 mL) in dichloromethane (25 mL) was added byaddition funnel slowly over 10 minutes until bubbling stopped.trans-4-Dibenzylaminocyclohexanol (17.14 g, 58.10 mmol) indichloromethane (150 mL) as then dripped in slowly. After 30 minutes,triethylamine (56 mL) was then added dropwise and then the solutionstirred at ambient temperature. The reaction was monitored via TLC toassure starting material consumption. The solution was then condensedunder reduced pressure and partitioned between water and ethyl acetate.The organics were dried over magnesium sulfate, filtered and solventremoved under reduced pressure. The resultant oil was purified viasilica gel chromatography (30% ethyl acetate/hexanes) to afford thetitle compound (13.71 g, 81%). ES-MS (m/z) 294 [M+1]⁺.

trans-4-Dibenzylamino-1-methylcyclohexanol

To a solution of trans-4-dibenzylaminocyclexanone (1.40 g, 4.77 mmol) inTHF at 0° C. (40 mL) was added a 3.0 M methylmagnesium bromide solutionon THF (6.36 mL, 19.1 mmol) dropwise. The solution was allowed to warmto ambient temperature and allowed to stir for 16 hours. The solutionwas quenched with saturated ammonium chloride solution and partitionedbetween water and ethyl acetate (three times). The organics werecombined, dried over magnesium sulfate, filtered and solvent removedunder reduced pressure. The resultant oil was purified via silica gelchromatography (15% ethyl acetate/hexanes) to afford the title compound(2.21 g, 17%). ES-MS (m/z) 310.6 [M+1]⁺.

trans-4-Amino-1-methylcyclohexanol

To a solution of trans-4-dibenzylamino-1-methylcyclohexanol (2.21 g,7.15 mmol) in ethanol (50 mL) was added palladium hydroxide (0.663 g,30% by wt.). The solution was flushed with fresh hydrogen gas andallowed to stir at ambient temperature for 16 hours. Starting materialconsumption was confirmed via LC-MS. The solution was filtered throughcelite and washed with additional ethyl acetate. The filtrate wascondensed under reduced pressure to afford the title compound(quantitative). ES-MS (m/z) 130.4 [M+1]⁺.

Example 5.38 Amide Coupling

Amides coupling reactions as set forth above can be accomplished byMethods A-C described below.

Method A: HATU

0.164 g (0.30 mmol) of A was dissolved in 5 ml of DMF and 0.140 g (1.2eq.) of HATU was added in one lot. The reaction was stirred at roomtemperature for about 0.5 h under a nitrogen atmosphere, 0.040 g (1.2eq.) of N-methylpiperazine was added and stirring continued overnight.The reaction mixture was purified using preparative chromatography usinga 15-40% gradient acetonitrile/water (0.1% TFA). After analyzingfractions by HPLC, pure fractions were combined and concentrated to theTFA salt. TFA was exchanged using 1N HCl and the TFA was extracted(10×10 ml) using ether. Upon neutralization of the aqueous layer, thefreebase crashed out and was collected and dried to give 0.020 g of B in10% yield.

Method B: HATU/HBTU

A solution of A (1 mmol) in 10 ml DMF (0.1 M) was treated with 1.2 eq.of HATU or HBTU (1.2 mmol) and stirred under a nitrogen atmosphere atroom temperature for about 0.5 h and 1.2 eq of N-methylpiperazine (1.2mmol) was added. The reaction mixture was stirred at room temperatureovernight. After concentrating the reaction mixture, it was purifiedusing preparative chromatography. The clean fractions were combined andconcentrated to the TFA salt. TFA was exchanged using 1N HCl and the TFAwas extracted with ether. Finally, the HCl salt was obtained uponconcentration of the aqueous layer.

Method C: HOBT/EDCI

A solution of A (1 mmol) in 10 ml DMF (0.1 M) was treated with 2.0 eq.of HOBT (2.0 mmol), 2.4 eq of EDCI (2.4 mmol), 2.4 eq of theN-methylpiperazine (2.4 mmol) and stirred under a nitrogen atmosphere atroom temperature overnight. After concentrating the reaction mixture, itwas purified using preparative chromatography. The clean fractions werecombined and concentrated to the TFA salt. TFA was exchanged using 1NHCl and the TFA was extracted with ether. Finally, the HCl salt wasobtained upon concentration of the aqueous layer.

Certain intermediates and reactants useful in the preparation of theaminopurine compounds can be prepared as described in Examples 5.15 to5.29, below.

Example 5.39 Electron-poor Anilines

The chloropyrimidine compound is dissolved in acetic acid and thecorresponding aniline is added. The reaction is stirred overnight atroom temperature. Water is added to the reaction mixture until aprecipitate forms. The precipitate is filtered out and dried under highvacuum.

Example 5.40 Acylation/Mesylation/Chloroformylation of Amines

An amine is suspended in methylene chloride and triethylamine is added.The mixture is stirred at room temperature until a clear solution isobtained. The corresponding acyl chloride, methanesulfonyl chloride ormethyl chloroformate is added and the reaction mixture is stirred forabout 2 h. Typically, mono and diacylated compounds are obtained. Thedesired monoacylated product is obtained in a pure form afterpurification using semi-preparative HPLC.

Example 5.41 cis-Ethyl-4-aminocyclohexanecarboxylate Hydrochloride

19.3 mL of concentrated hydrochloric acid (2.8 eq) was added to asolution of cis-4-aminocyclohexane carboxylic acid (10 g, 69.83 mmol) inanhydrous ethyl alcohol (250 mL). Mixture was stirred overnight at about60° C. and then cooled to room temperature. Solvent was evaporated invacuo. Crude material was then redissolved in acetonitrile, sonicated,and concentrated to a solid in vacuo. This acetonitrile wash wasrepeated three times to obtain 11.5 g of while solid (96% yield).Trans-ethyl 4-aminocyclohexanecarboxylate hydrochloride can be preparedfollowing the same procedure using trans-4-aminocyclohexane carboxylicacid.

Example 5.42 Ester Hydrolysis (Basic Conditions)

The appropriate ester is added to a solution of 10 equivalent of LiOH in1:1 THF/H₂O. Gradually, the reaction mixture is heated to about 60° C.and stirred overnight. After about 12 h, the presence of the desiredcompound is verified via LC/MS. The reaction mixture is concentrated and1N HCl is added dropwise. The aqueous layers are extracted with2-butanone (3×100 ml) and dried with MgSO₄. After filtering off theMgSO₄, the compound is concentrated under reduced pressure and purifiedusing column chromatography or reverse-phase HPLC.

Example 5.43 Ester Hydrolysis (Acidic Conditions)

Carboxylic acid ethyl ester is dissolved in 2N hydrochloric acid. Theresulting solution is heated to about 75° C. and stirred for about threehours. After cooling to room temperature, excess aqueous ammoniumhydroxide is added and the solvent is evaporated under reduced pressure.Trituration of the residue with ethanol, followed by filtration, givesthe corresponding carboxylic acid.

Example 5.44 Carboxamide Formation

Oxalyl chloride is added, under an N_(2 (g)) atmosphere, dropwise to asolution of the appropriate carboxylic acid in DCM. DMF is then added tothe solution and bubbling is observed. After about 6 h, the reactionmixture is concentrated under reduced pressure and DCM and NH₄OH (conc)are added. The reaction mixture is stirred for about an additional 4 hbefore being concentrated and purified via reverse-phase preparativeHPLC (20-80% acetonitrile/water (0.1% TFA)).

Example 5.45 trans-(4-Aminocyclohexyl)methan-1-ol

Trans-4-aminocyclohexane carboxylic acid hydrochloride (2.00 g, 14.3mmol) was added in small portions to a stirred, hot (70-85° C.) solutionof Red-Al (27.0 g) for 2 h (a semisolid formed), and heating wascontinued overnight. After 24 h, the reaction mixture was cooled to roomtemperature and treated with a solution of NaOH (3.8 g) in H₂O (34 ml).Following the addition, the reaction was gradually heated to 80° C., andcooled. The toluene layer was separated and the aqueous layer wasextracted with CHCl₃ (3×100 ml). The organic layers were dried withMgSO₄ and then concentrated under reduced pressure to provide thedesired compound (1.81 g, 14.0 mmol) as a pure white solid in 50% yield.ES-MS: 130 (M+1).

Example 5.46 trans-2-(4-Aminocyclohexyl)propan-2-ol

trans-Phenylmethyl 4-[N,N-dibenzylamino]cyclohexanecarboxylate

To an 80° C. heated mixture of trans-4-Aminocyclohexanecarboxylic acid(8 g, 55.97 mmol) and K₂C03 (23.4 g) in 112 mL of CH₃CN, was addeddropwise a solution of BnBr (23.3 mL, 195.5 mmol) in 70 mL of CH₃CN byaddition funnel. Reaction stirred overnight at 80° C. The reaction wascooled to room temperature and filtered. Precipitate did not form infiltrate like it did with the cis-isomer so the filtrate wasconcentrated to an oil and carried on to next step (23.01 g, 99% yield).

trans-2-{4-[N,N-Dibisbenzylamino]cyclohexyl}propan-2-ol

Phenylmethyl 4-[bisbenzylamino]cyclohexanecarboxylate (6 g, 14.50 mmol)was combined with 460 mL of THF, flushed with N₂ and cooled to 0° C.Methylmagnesium bromide (48 ml, 145.08 mmol) was added to reaction andleft to stir overnight. The reaction was quenched with 600 mL ofsaturated NH₄Cl. Layers were separated and organics were washed withsaturated NaHCO₃ and brine (100 mL). Organics were dried with Na₂SO₄ andconcentrated in vacuo. The mixture was dried overnight on high vacuum toafford 3.89 g of solid product (80% yield).

trans-2-(4-Aminocyclohexyl)propan-2-ol

3.85 g of trans-2-{4-[N,N-dibenzylamino]cyclohexyl}propan-2-ol (11.40mmol) was combined with 3.3 g of 20 wt % palladium hydroxide anddissolved with 100 mL of anhydrous ethyl alcohol. The mixture wasflushed with H₂ (4×) before a H₂ balloon was inserted into the reactionand allowed to stir overnight. N₂ was bubbled through for about 20minutes and the catalyst was filtered off. The reaction was washed withmethanol. The filtrate was concentrated in vacuo and redissolved inacetonitrile and sonicated which produced a white solid. The mixture wasfiltered and 0.67 g of white product was obtained (37% yield).

Example 5.47 trans-4-(N,N-Dibenzylamino)cyclohexanol

To a 1-L round bottom flask equipped with magnetic stirring, nitrogeninlet and dropping funnel was charged trans-4-aminocyclohexanolhydrochloride (50.0 g, 0.33 mol), sodium carbonate (139.9 g, 1.32 mol)and anhydrous DMF (400 mL). Stirring was initiated and benzyl bromide(82.3 mL, 0.69 mol) was added via dropping funnel over a period of about15 minutes. A slight exotherm was observed following addition of benzylbromide. The reaction was allowed to stir at ambient temperature forabout 18 h, then a sample was taken for LCMS analysis. LCMS indicatedcomplete conversion of starting material at this time.

The reaction mixture was filtered through a medium frit under vacuum toremove salts, and the filtrate was diluted with water (400 mL) and MTBE(400 mL). The mixture was agitated in a separatory funnel, then thelower aqueous layer was drained off. The organic layer was decanted,then the aqueous layer was extracted with MTBE (200 mL). The organiclayers were combined and extracted twice with water (300 mL), thenextracted with saturated brine (100 mL). The organic layer was dried(sodium sulfate), filtered and evaporated under reduce pressure to yielda white solid. The solid was further dried at 50° C. under vacuum. Tothe dry solid was added cyclohexane (400 mL) and the mixture was stirredin a water bath at 80° C. until most solids were in solution. Thesolution was quickly filtered using celite and a fritted funnel undervacuum. The resulting slurry was heated to boiling to redissolve solidsand stirred while cooling slowly. The fine white crystals were filteredon a frit, then washed with two portions of cyclohexane (50 mL). Thecrystals were then dried for about 18 h at 60° C. under vacuum to yield58.4 g (60%) of pure material. LRMS (ES) m/e 296.2 [MH]⁺; HPLC (5→70%acetonitrile/water (0.1% TFA) over 20 minutes) RT=9.55 min.

Example 5.48trans-N,N-Dibenzyl-4-(2-(piperidin-1-yl)ethoxy)cyclohexanamine

Into a nitrogen flushed 250-mL round bottom flask was charged 35%potassium hydride suspension in oil (16.27 g, 142 mmol) and hexanes (60mL). The mixture was stirred briefly, then allowed to settle. Thesupernatant was drawn off via syringe, thentrans-4-(dibenzylamino)cyclohexanol (10.0 g, 33.9 mmol),1-(2-chloroethyl)piperidine hydrochloride (18.72 g, 101.7 mmol) anddioxane (120 mL) were added and the mixture stirred at ambienttemperature. The reaction mixture tends to thicken. Once hydrogenevolution had ceased the mixture was brought to 90-100° C. for about 2h, then cooled to ambient temperature. Methanol (20 mL) was added andthe mixture was stirred until hydrogen evolution ceased. The solventswere evaporated under reduced pressure, and the residue partitionedbetween 5% sodium carbonate solution (100 mL) and dichloromethane (200mL). The layers were separated and the aqueous layer extracted withdichloromethane (100 mL). The organic extracts were combined and dried(sodium sulfate), filtered and evaporated under reduced pressure. Theresidue was chromatographed (silica gel, 330 g, using a gradient ofchloroform-ethanol-conc. ammonia soln. from (98:2:0) to (92:8:2)) togive 4.7 g of an oil (61%). LRMS (ES) m/e 407.3 [MH]⁺; HPLC (5→70%acetonitrile/water (0.1% TFA) over 20 minutes) RT=9.23 min.

Example 5.49 trans-4-(2-(Piperidin-1-yl)ethoxy)cyclohexanamine

Trans-N,N-dibenzyl-4-(2-(piperidin-1-yl)ethoxy)cyclohexanamine (4.7 g,11.6 mmol), 20% Pd(OH)₂/C (0.94 g) and methanol (40 mL) were chargedinto a septum-sealed flask. The reaction mixture was placed underballoon pressure of hydrogen for 18 h at ambient temperature, at whichtime LCMS indicated complete debenzylation to form the free amine. Thecatalyst was filtered off and the filtrate evaporated under reducedpressure to give 2.42 g of a crystalline material (93%). In some casesan additional portion of catalyst (ca. 50% of the initial charge) wasneeded in order to attain complete reaction. LRMS (ES) m/e 227.2 [MH]⁺;¹H NMR (300 MHz, CD₃OD) δ 3.51 (t, 2H), 3.12 (m, 1H), 2.61 (m, 1H), 2.42(t, 2H), 2.18 (m, 4H), 1.93 (m, 2H), 1.80 (m, 2H), 1.50 (m, 4H), 1.33(m, 2H), 1.12 (m, 4H).

Example 5.50 1-Methylsulfonylpyrrolidin-3S-amine Hydrochloride

(3S)-3-(tert-Butylcarbonylamino)pyrrolidine (10.0 mmol, 1 eq.) andN,N-diisopropylethylamine (25.0 mmol, 2.5 eq.) were dissolved in 20 mlDCM. Methanesulfonylchloride (10.0 mmol, 1 eq.) was added dropwise andthe reaction mixture was stirred overnight at rt. After adding water thephases were separated and the organic phase was dried over MgSO₄ andevaporated to give the desired product. This compound was dissolved in12 ml dioxane and 23 ml 4N HCl in dioxane was added, The reactionmixture was stirred overnight. Evaporation of the solvent and additionalcoevaporation with toluene gave the desired product. This reaction canalso be performed with the R-enantiomer.

Example 5.51 cis-4-[(2-piperidylethoxy)methyl]cyclohexylamine

Benzyl cis-4-[N,N-dibenzylamino]cyclohexane carboxylate

cis-4-Aminocyclohexanecarboxylic acid (10.0 g, 67.48 mmol) was dissolvedin 140 mL of dry acetonitrile. Solid potassium carbonate (28.0 g, 202.6mmol) was added. The suspension was heated to about 80° C. To thissolution, was added benzyl bromide (28.09 mL, 236.2 mmol) in 70 mL ofacetonitrile, dropwise via addition funnel. The reaction mixture wasstirred at about 80° C. under nitrogen for about 2 hours then about 40°C. overnight. The reaction was cooled to room temperature and thesuspension was filtered. The filtrate was concentrated under reducedpressure. The compound was purified using column chromatography onsilica gel (100% hexanes to remove excess benzyl bromide, then 10% ethylacetate in hexanes). The product was isolated as a white solid (14.35 g,51% yield): ES-MS (m/z) 414.

cis-{4-[N,N-Dibenzylamino]cyclohexyl}methan-1-ol

A solution of benzyl cis-4-[N,N-dibenzylamino]cyclohexane carboxylate(14.35 g, 34.70 mmol) in dry THF (180 mL) was prepared and then cooleddown to −78° C. A solution of lithium aluminum hydride (104.0 mL, 1.0 Msolution in diethyl ether) was added dropwise. At the end of theaddition, the reaction temperature was raised to about −50° C.(acetonitrile/dry ice) and the temperature was maintained for about 3hours. The completion of the reaction was monitored by LC-MS. Thereaction was quenched by dropwise addition of saturated aqueous sodiumsulfate. Saturated aqueous sodium bicarbonate (10 mL) and diethyl ether(50 mL) were then added. A white solid formed and was removed byfiltration and washed with THF. The organic phase was separated andconcentrated under reduced pressure. The product was purified by slowprecipitation. The residue was dissolved in 5 mL of diethyl ether andthe solution was layered with 50 mL of hexanes. Clear large crystalswere obtained after overnight diffusion. (7.279 g, 67% yield) ES-MS(m/z) 310.

cis-N,N-Dibenzyl-N-{4-[(2-piperidylethoxy)methyl]cyclohexyl}amine

cis-{4-[N,N-Dibenzylamino]cyclohexyl}methan-1-ol (2.851 g, 9.21 mmol)and (2-chloroethyl)piperidine hydrochloride were suspended in 50 mL ofdioxane. Potassium hydride (3.16 g, 35% by weight in mineral oil) wasadded dropwise in suspension in 20 mL of dioxane. The reaction mixturewas stirred at room temperature for about 1 hour. The reaction mixturewas then warmed to about 70° C. and one equivalent of potassium hydride(1.05 g, 35% by weight in mineral oil) was added dropwise. Thetemperature was maintained for about 2 hours, after which the conversionwas complete. The reaction was cooled to room temperature and quenchedwith methanol. Solvents were removed under reduced pressure.Acetonitrile was added (200 mL) and the gray brown solid was removed byfiltration. The crude was purified by column chromatography on silicagel using 3% (ethanol/ammonium hydroxide=8:1) in dichloromethane. Theproduct was isolated as an orange oil that solidified under vacuum.(2.84 g, 72% yield): ES-MS (m/z) 421.

cis-4-[(2-Piperidylethoxy)methyl]cyclohexylamine

cis-N,N-Dibenzyl-N-{4-[(2-piperidylethoxy)methyl]cyclohexyl}amine (2.84g, 6.65 mmol) was dissolved in 20 mL of ethanol. Palladium hydroxide(20% weight) was added (50 mg) and the reaction was stirred overnightunder an atmosphere of hydrogen. The catalyst was removed by filtrationand washed with small portions of ethanol. The filtrate was concentratedand used without further purification. (quantitative yield): ES-MS (m/z)241.

Example 5.52 cis-4-(Methoxymethyl)cyclohexyl amine

cis-4-[(tert-Butoxy)carbonylamino]cyclohexane carboxylic acid

cis-4-Aminocyclohexyl carboxylic acid (2.0 g, 13.96 mmol) was dissolvedin 40 mL of 1,4-dioxane. Two equivalents of di-tert-butyl-dicarbonate(6.094 g, 27.92 mmol) were added followed by 3 equivalents of sodiumbicarbonate (4.06 g, 41.88 mmol) dissolved in 40 mL of water. Thereaction mixture was stirred at room temperature for about 12 hours. Thecompletion of the reaction was monitored by LC-MS. Saturated aqueousKHSO₄ was added dropwise, until gas evolution stopped. The solvent wasthen removed under reduced pressure and the crude product was extractedin ethyl acetate. The combined organic extracts were washed with aqueoussaturated KHSO₄ and dried over Na₂SO₄. The solvent was removed underreduced pressure, yielding 2.6 g of product. Based on ¹H NMR, theproduct was pure and used in subsequent steps without furtherpurification ES-MS (m/z) 244.

cis-(tert-Butoxy)-N-[4-(hydroxymethyl)cyclohexyl]carboxamide

cis-4-[(tert-Butoxy)carbonylamino]cyclohexane carboxylic acid (2.6 g,10.68 mmol) was dissolved in THF (20 mL) and cooled to −10° C.(MeOH-ice). N-Methyl morpholine was added followed by isobutylchloroformate (1.175 mL, 10.68 mmol). After 10 min, NaBH₄ was added as asolid in one portion (1.213 g, 32.06 mmol). The reaction mixture waswarmed to 0° C. and methanol was added dropwise (13.35 mL). After about30 min, the reaction was quenched with 5% aqueous KHSO₄. The reactionmonitored by LC-MS was complete. The crude product was extracted withethyl acetate and the combined extracts were dried over Na₂SO₄. Acolorless oil was obtained and solidified slowly at room temperature.The product and purity were assessed by LC-MS and ¹H NMR. No furtherpurification was necessary. (quantitative yield) ES-MS (m/z) 230.

cis-4-(Methoxymethyl)cyclohexyl amine

Sodium hydride (72 mg, 1.78 mmol, 60% by weight suspended in mineraloil) was washed three times with 10 mL portions of hexanes, andsuspended in dry THF (12 mL). The suspension was cooled to 0° C. To thissuspension, cis-(tert-butoxy)-N-[4-(hydroxymethyl)cyclohexyl]carboxamide(0.273 g, 1.20 mmol) and 15-crown-5 (0.250 mL, 1.25 mmol) were added.The reaction mixture was then stirred at 0° C. for about 30 min. Methyliodide was then added dropwise (75 μL, 1.20 mmol). Since the reactionwas not complete after overnight stirring at room temperature, themixture was cooled to 0° C. and reacted with 100 mg of sodium hydrideand 0.250 mL of 15-crown-5. After about 2 hours at room temperature, thereaction was complete. The reaction was quenched by the slow addition ofwater and the crude product was extracted with ethyl acetate.Purification was effected by column chromatography on silica gel using20% ethyl acetate in hexanes as the eluent. ES-MS (m/z) 244.cis-(Tert-butoxy)-N-[4-(methoxymethyl)cyclohexyl]carboxamide wasdissolved in ethanol (5 mL) and the solution was treated with 1 mL ofacetyl chloride at room temperature. The reaction mixture was stirred atroom temperature overnight. The solvent was removed under reducedpressure and the resulting solid was used without further purification.(79% yield) ES-MS (m/z) 144.

Example 5.53 trans-4-Methoxycyclohexylamine

trans-(tert-Butoxy)-N-(4-methoxycyclohexyl)carboxamide

Sodium hydride (60% in mineral oil, 278 mg, 6.96 mmol) was suspended inTHF (5 mL) and cooled to 0° C.trans-tert-Butoxy-N-(4-hydroxycyclohexyl)carboxamide (1 g, 4.64 mmol)and 15-crown-5 (0.965 mL, 4.88 mmol) were added and the reaction mixturewas stirred at 0° C. for about 30 minutes. Iodomethane (0.289 mL, 4.64mmol) was added and the reaction stirred at 0° C. for about 1 hour afterwhich the LCMS showed it was complete. The reaction was quenched withmethanol, the solvents removed in vacuo and the crude purified by columnchromatography (SiO₂, 8:2 n-hexanes/ethyl acetate) to afford 642 mg ofthe methyl ether. ES-MS: 230 (M+1).

trans-4-Methoxycyclohexylamine

trans-(tert-Butoxy)-N-(4-methoxycyclohexyl)carboxamide (642 mg, 2.80mmol) was dissolved in ethanol (5 mL) and cooled to 0° C. Acetylchloride (1.5 mL) was added and the reaction was allowed to reach roomtemperature and stirred overnight. Solvent was removed in vacuo to givethe desired product (458 mg, quantitative yield) as a hydrochloridesalt. ES-MS: 130 (M+1).

The Aminopurine Compounds can be assayed for their activity according tothe following procedures.

JNK1 Assay

To 10 μL of an Aminopurine Compound in 20% DMSO/80% dilution bufferconsisting of 20 mM HEPES (pH 7.6), 0.1 mM EDTA, 2.5 mM magnesiumchloride, 0.004% Triton ×100, 2 μg/mL leupeptin, 20 mMβ-glycerolphosphate, 0.1 mM sodium vanadate, and 2 mM DTT in water isadded 30 μL of 50 ng His6-JNK1 in the same dilution buffer. The mixtureis preincubated for 30 minutes at room temperature. Sixty microliter of10 μg GST-c-Jun(1-79) in assay buffer consisting of 20 mM HEPES (pH7.6), 50 mM sodium chloride, 0.1 mM EDTA, 24 mM magnesium chloride, 1 mMDTT, 25 mM PNPP, 0.05% Triton ×100, 11 μM ATP, and 0.5 μCi γ-³²P ATP inwater is added and the reaction is allowed to proceed for 1 hour at roomtemperature. The c-Jun phosphorylation is terminated by addition of 150μL of 12.5% trichloroacetic acid. After 30 minutes, the precipitate isharvested onto a filter plate, diluted with 50 μL of the scintillationfluid and quantified by a counter. The IC₅₀ values are calculated as theconcentration of the Aminopurine Compound at which the c-Junphosphorylation is reduced to 50% of the control value. Certaincompounds have an IC₅₀ value ranging from 0.01-10 μM in this assay.

JNK2 Assay

To 10 μL of an Aminopurine Compound in 20% DMSO/80% dilution bufferconsisting of 20 mM HEPES (pH 7.6), 0.1 mM EDTA, 2.5 mM magnesiumchloride, 0.004% Triton ×100, 2 μg/mL leupeptin, 20 mMβ-glycerolphosphate, 0.1 mM sodium vanadate, and 2 mM DTT in water isadded 30 μL of 50 ng His6-JNK2 in the same dilution buffer. The mixtureis preincubated for 30 minutes at room temperature. Sixty microliter of10 μg GST-c-Jun(1-79) in assay buffer consisting of 20 mM HEPES (pH7.6), 50 mM sodium chloride, 0.1 mM EDTA, 24 mM magnesium chloride, 1 mMDTT, 25 mM PNPP, 0.05% Triton ×100, 11 μM ATP, and 0.5 μCi γ-³²P ATP inwater is added and the reaction is allowed to proceed for 1 hour at roomtemperature. The c-Jun phosphorylation is terminated by addition of 150μL of 12.5% trichloroacetic acid. After 30 minutes, the precipitate isharvested onto a filter plate, diluted with 50 μL of the scintillationfluid and quantified by a counter. The IC₅₀ values are calculated as theconcentration of the Aminopurine Compound at which the c-Junphosphorylation is reduced to 50% of the control value. Certaincompounds have an IC₅₀ value ranging from 0.01-10 μM in this assay.

JNK3 Assay

To 10 μL of an Aminopurine Compound in 20% DMSO/80% dilution bufferconsisting of 20 mM HEPES (pH 7.6), 0.1 mM EDTA, 2.5 mM magnesiumchloride, 0.004% Triton ×100, 2 μg/mL leupeptin, 20 mMβ-glycerolphosphate, 0.1 mM sodium vanadate, and 2 mM DTT in water isadded 30 μL of 200 ng His6-JNK3 in the same dilution buffer. The mixtureis preincubated for 30 minutes at room temperature. Sixty microliter of10 μg GST-c-Jun(1-79) in assay buffer consisting of 20 mM HEPES (pH7.6), 50 mM sodium chloride, 0.1 mM EDTA, 24 mM magnesium chloride, 1 mMDTT, 25 mM PNPP, 0.05% Triton ×100, 11 μM ATP, and 0.5 μCi γ-³²P ATP inwater is added and the reaction is allowed to proceed for 1 hour at roomtemperature. The c-Jun phosphorylation is terminated by addition of 150μL of 12.5% trichloroacetic acid. After 30 minutes, the precipitate isharvested onto a filter plate, diluted with 50 μL of the scintillationfluid and quantified by a counter. The IC₅₀ values are calculated as theconcentration of the Aminopurine Compound at which the c-Junphosphorylation is reduced to 50% of the control value. Certaincompounds have an IC₅₀ value ranging from 0.001-10 μM in this assay.

p38α Assay

The p38α kinase assay is carried out in 96-well plate format at a finalvolume of 100 μl. ATP is used at a final concentration of 340 μM, threefold the apparent K_(m). Kinase is diluted in Dilution Buffer (20 mMHEPES pH 7.6, 0.1 mM EDTA, 2.5 mM MgCl₂, 0.004% (w/v) Triton ×100, 2μg/ml Leupeptin, 20 mM B-glycerol phosphate, 0.1 mM Na₃VO₄, 2 mMdithiothreitol) and pre-mixed with MBP diluted in Substrate SolutionBuffer (20 mM HEPES pH 7.6, 50 mM NaCl, 0.1 mM EDTA, 2.5 mM MgCl₂, 0.05%(w/v) Triton ×100) to give final assay concentrations of 50 ng/well (7.8nM) for p38α and 30 μg/well (16 μM, 2×K_(m)) for MBP. The p38α/MBP mix(85 μl) is added to an Aminopurine Compound (5 μl) diluted in 100% DMSOto give a final DMSO assay concentration of 5% (v/v). Enzyme, substrateand Aminopurine Compound are allowed to equilibrate at room temperaturefor about 15 minutes. The reaction is started by addition of 10 μl10×ATP in kinase buffer (130 mM MgCl₂ 6 mM dithiothreitol, 150 mMpara-nitrophenyl phosphate, 100 μCi/ml γ-[³³P]-ATP). Reactions areallowed to proceed for 60 minutes before precipitation of protein viatrichloroacetic acid (7.2% TCA final). After a 30 minute incubation withTCA, reaction products are collected onto glass microfilter 96-wellplates (Millipore MAHF CIH60) using a Packard Filtermate. Theprecipitate is washed with Phosphate Buffered Saline and the amount ofphosphate incorporated into MBP is quantified by scintillation countingusing a Packard Topcount-NXT.

Jurkat T-Cell Il-2 Production Assay

Jurkat T cells (clone E6-1) are purchased from the American TissueCulture Collection and maintained in growth media consisting of RPMI1640 medium containing 2 mM L-glutamine (Mediatech), with 10% fetalbovine serum (Hyclone) and penicillin/streptomycin. All cells arecultured at 37° C. in 95% air and 5% CO₂. Cells are plated at a densityof 0.2×10⁶ cells per well in 200 μL of media. Aminopurine Compound stock(20 mM) is diluted in growth media and added to each well as a 10×concentrated solution in a volume of 25 μL, mixed, and allowed topre-incubate with cells for 30 minutes. The compound vehicle(dimethylsulfoxide) is maintained at a final concentration of 0.5% inall samples. After 30 minutes the cells are activated with PHA (phorbolmyristate acetate; final concentration 50 μg/mL) and PHA(phytohaemagglutinin; final concentration 2 μg/mL). PMA and PHA areadded as a 10× concentrated solution made up in growth media and addedin a volume of 25 μL per well. Cell plates are cultured for 10 hours.Cells are pelleted by centrifugation and the media removed and stored at−20° C. Media aliquots are analyzed by sandwich ELISA for the presenceof IL-2 as per the manufacturers instructions (Endogen). The IC₅₀ valuesare calculated as the concentration of the Aminopurine Compound at whichthe Il-2 production was reduced to 50% of the control value. Certaincompounds have an IC₅₀ value ranging from 0.01-10 μM in this assay.

Rat In Vivo LPS-Induced TNF-α Production Assay

Male CD rats procured from Charles River Laboratories at 7 weeks of ageare allowed to acclimate for one week prior to use. A lateral tail veinis cannulated percutaneously with a 22-gage over-the-needle catheterunder brief isoflurane anesthesia. Rats are administered an AminopurineCompound either by intravenous injection via the tail vein catheter ororal gavage 15 to 180 min prior to injection of 0.05 mg/kg LPS (E. Coli055:BS). Catheters are flushed with 2.5 mL/kg of normal injectablesaline. Blood is collected via cardiac puncture 90 minutes after LPSchallenge. Plasma is prepared using lithium heparin separation tubes andfrozen at −80° C. until analyzed. TNF-α levels are determined using arat specific TNF-α ELISA kit (Biosource). The ED₅₀ values are calculatedas the dose of the Aminopurine Compound at which the TNF-α production isreduced to 50% of the control value. Certain compounds have an ED₅₀value ranging from 1-30 mg/kg in this assay.

Abl LANCE HTRF Tyrosine Kinase Assay

The day prior to performing the assay, the following are prepared:

(1) 2 mg/ml BSA/0.4% Triton ×100/50 mM HEPES pH 7.6 (kept at 4° C.);

(2) Streptavidin-APC (PerkinElmer Life Sciences CR130-100) diluted innH₂0 according to instructions (kept at 4° C., up to 2 weeks maximum);

(3) Tyrosine Kinase Biotinylated Peptide Substrate 2 (Pierce 29914)diluted in nH₂0 (kept at 4°);

(4) Aminopurine Compound dilutions in DMSO.

The following mixtures are prepared the day on which the assay isperformed:

(5) 2 mM DTT/50 mM HEPES pH 7.6;

(6) 2 mM Staurosporine for Background Control and 1:3 serial dilutionsfor Reference Control in DMSO;

(7) LANCE Mixture in 2 mg/ml BSA/0.2% Triton ×100/50 mM HEPES pH 7.6prepared as follows: 250 nM Streptavidin-APC (PerkinElmer Life SciencesCR130-100), 250 nM Tyrosine Kinase Biotinylated Peptide Substrate 2(Pierce 29914), and 250 ng/ml Eu-anti-phosphoTyrosine (PerkinElmer LifeSciences AD0066);

(8) Kinase/detection mixture prepared as follows: 18.7 ng/ml Abl(Calbiochem 102555), 5.9 mM MgCl₂, and 58.8% LANCE Mixture from (7),brought to final volume with 2 mM DTT/50 mM HEPES pH 7.6;

(9) 240 μM ATP in 2 mM DTT/25 mM HEPES pH 7.4.

To a black 384 well microtiter plate (Corning 3710) is added 2.5 μl/wellcompound dilutions/DMSO and 42.5 μl/well kinase/detection mixture. Theplate is incubated for 5 minutes on shaker followed by 10 minutes staticincubation at room temperature.

5 μl/well ATP is added to the plate and the plate is incubated for 5minutes on shaker followed by 55 minutes static incubation at roomtemperature.

30 μl/well 16.7 mM EDTA is added to the plate and the plate is incubatedfor at least 2 minutes on a shaker followed by 30 minutes staticincubation at room temperature. The plate is then read (TR-FRET) onPackard Fusion instrument.

Certain compounds have an IC₅₀ value ranging from 0.01-10 μM in thisassay.

Alamar Blue Assay for K562 Cells

Chronic myelogenous leukemia K562 is routinely maintained in RPMI 1640with 10% heat inactivated FBS and 1% Penicillin-Streptomycin. For cellproliferation assay, K562 cells are plated in 96-well round bottomplates. Cells are treated with an Aminopurine Compound the same day ofplating. For dose response experiments, a 30 mM solution of anAminopurine Compound is diluted to give final concentrations of 30 μM, 3μM, 0.3 μM, 0.03 μM, and 0.003 μM. The final DMSO concentration is 0.2%in each well. Alamar Blue is used to quantify cell number after a 72hour incubation with an Aminopurine Compound. Certain compounds have anIC₅₀ value ranging from 0.1-10 μM in this assay.

Ozone-Induced Effects in Mice

The following protocol can be used to assay an Aminopurine Compound foractivity against ozone-induced effects in vivo.

Mice: Pathogen free 6-8 week old male BALB/c mice (Harlan, UK) arehoused within ‘maximiser’ filter-topped cages (Maximiser, Theseus cagingsystem Inc., Hazelton, Pa., USA).

Aminopurine Compound: The Aminopurine Compound is prepared as a 30 mg/kgsolution in a vehicle of 10% Ethanol, 15% Cremophor-El, 30% PolyethyleneGlycol-400 and 20% Propylene Glycol (Sigma, Dorset, UK) in sterilesaline. Mice are injected intraperitoneally with the AminopurineCompound (30 mg/kg; 0.16 ml volume). Control animals receive vehiclealone, in the same volume as the Aminopurine Compound.

Aminopurine Compound Administration Protocol: The effect of theAminopurine Compound or of vehicle alone in air or ozone-exposed mice isstudied at two time-points. Three hours following exposure, lung tissuesare collected for Affymetrix microarray, real-time polymerase chainreaction, Western blot analysis and bronchioalveolar (BAL) areperformed. Twenty to twenty-four hours following exposure, lungresistance (R_(L)) is measured and BAL is performed. Mice are exposed toozone produced by an ozonizer (Model 500 Sander Ozoniser, Germany),mixed with air for 3 h at a concentration of 3 parts per million (ppm)within a sealed Perspex container. Ozone concentration is continuallymonitored with an ozone probe (ATi Technologies, Oldham, UK). Controlanimals receive medical air only. Mice receive either the AminopurineCompound or vehicle two hours prior to air- or ozone-exposure, eighthours after exposure and two hours prior to measurement of R_(L).

Measurement of Lung Resistance (R_(L)): Twenty-four hours followingexposure, mice are anesthetized with an intraperitoneal injection ofanesthetic solution containing midazolam (Roche Products Ltd., WelwynGarden City, UK) and Hypnorm (0.315 mg/ml fentanyl citrate and 10 mg/mlfluanisone; Janssen Animal Health, Wantage, UK). Mice aretracheostomised and ventilated (Mini Vent type 845, Hugo SachElectronic, Germany; rate: 250 breaths/min and tidal volume: 250 μl).Mice are monitored in a whole body plethysmograph with apneumotachograph connected to a transducer (EMMS, Hants, UK).Transpulmonary pressure is assessed via an esophageal catheter (EMMS,Hants, UK). Instantaneous calculation of pulmonary resistance (R_(L)) isobtained. Increasing concentrations of acetylcholine chloride (ACh)(Sigma, Dorset, UK) (4-256 mg/ml) are administered with an ultrasonicnebulizer, and R_(L) is recorded for a 5-min period following eachconcentration. R_(L) after each concentration is expressed as percentagechange from baseline R_(L) measured following nebulized phosphatebuffered saline (PBS) (Sigma, Dorset, UK). The concentration ofacetylcholine required to increase R_(L) by 150% from baseline iscalculated (PC₁₅₀).

Bronchioalveolar Lavage: A previously-published method is used (Nath P.et al., European Journal of Pharmacology 506: 273-283 (2005)). Briefly,following an overdose of anaesthetic, mice are lavaged with a six 0.5 mlaliquots of PBS via the endrotracheal tube, and retrieved as thebronchioalveolar lavage fluid. Total cell counts and differential cellcounts from cytospin preparations stained by May-Grunwald-Giemsa stainare determined under an optical microscope (Olympus BH2, Olympus OpticalCompany Ltd., Tokyo, Japan). At least 400 cells are counted per mouseand identified as macrophages, eosinophils, lymphocytes and neutrophilsaccording to standard morphology under x400 magnification.

DNA Microarray: Total RNA is extracted from frozen lung tissue usingTriZol reagent (GIBCO, Paisley, UK) followed by RNeasy columns (Qiagen,Crawley, UK) for additional purity. Quantitation and quality assessmentof the RNA preparations are performed on a spectrophotometer (NanodropTechnologies, Wilmington, Del., USA) and Agilent Bioanalyzer (FosterCity, Calif., USA), respectively. Biotinylated antisense-RNA isgenerated with the MessageAmp II Kit (Ambion, Austin, Tex., USA).Briefly, first strand cDNA is synthesized from 1 μg total RNA withT7-oligo(dT) primers for 2 hours at 42° C. Second-strand cDNA issynthesized at 16° C. for 2 hours and then purified on Ambion cDNAfilter cartridges. In vitro transcription is conducted at 37° C. for 14hours with T7 RNA polymerase and biotinylated CTP and UTP (Enzo,Farmingdale, N.Y., USA). The biotinylated antisense-RNA is purified onfilter cartridges (Ambion, Tex., USA) and fragmented at 94° C. for 30minutes prior to use on DNA Microarray. Fragmented antisense-RNA (10 g)is hybridized to Affymetrix 430A v.2 mouse GeneChips for 16 hrs at 45°C. according to manufacturer's instructions (Affymetrix, Calif., USA).Mouse GeneChips are washed on the Fluidics Station and imaged on ahigh-resolution Scanner 3000 (Affymetrix, Calif., USA). The aboveprocedures are performed in duplicate for each sample. Gene expressiondata analysis is performed using GeneSpring software version 7.2(Agilent, Palo Alto, Pa., USA). Data normalization includes thefollowing steps, as recommended by Affymetrix: (i) values below 0.01 areset to 0.01 (ii) each gene measurement is divided by the 50th percentileof all measurements in that sample (iii) each gene measurement isdivided by the median of its measurements in all samples. Fold-changeanalysis is performed on average values from each pair of replicates.Clustering and generation of a condition tree are performed using theStandard Correlation parameter within GeneSpring.

cDNA synthesis, reverse transcription and real-time polymerase chainreaction (PCR): RNA extracted from each sample in the previous step isused for the following experiment. RNA (5 μg per sample) is used tosynthesize single-stranded complementary DNA (cDNA) using randomhexamers and an avian myeloblastosis virus reverse transcriptase(Promega, Southampton, UK). The cDNA generated is used as a template insubsequent real-time PCR analyses. Transcript levels are determined byreal-time PCR (Rotor Gene 3000, Corbett Research, Sydney, AUS) usingSYBR Green PCR Master Mix Reagent (Qiagen, Crawley, UK). Cyclingconditions are as follows: step 1: 15 min at 95° C.; step 2: 20 s at 94°C.; step 3: 20 s at 60° C.; step 4: 20 s at 72° C., with step 2 to step4 repeated 45 times. The standard curves used to determine relativeexpression for each primer are obtained by running real-time PCR for adiluted sample, for example, to 1:1, 1:10, 1:100 and 1:1000. Geneexpression is normalized to GAPDH.

Measurement of JNK activity in lung tissue: JNK activity is measured inthe lung tissue, using a previously-published method (Eynott P. R. etal., Immunology 112: 446-453 (2004). Briefly, 30 μg of total lungprotein per lane are separated through 12% denaturing-polyacrylamidegels and transferred to nitrocellulose membranes. The membranes areblocked with 5% non-fat dry milk in the following buffer (Tris 20 mM, pH7.6, NaCl 140 mM and 0.1% Tween) and then incubated overnight withaffinity-purified rabbit polyclonal antibodies, anti-non-phosphorylatedc-jun anti-phosphoserine-63 (Cell Signaling Technology, Beverly, Mass.,USA) and anti-phosphorylated c-jun (p-c-jun) (Cell Signaling Technology)as markers of JNK activity. Horseradish peroxidase-conjugatedanti-rabbit (diluted to 1:2,000 from Cell Signalling Technology) is usedas a secondary antibody and Enhanced Chemiluminescence (Amersham, Bucks,UK) reagent is used for detection. The bands, which are visualised byautoradiography, are quantified using a densitometer with Grab-It andGelWorks software (UVP, Cambridge, UK).

Data analysis: Data are presented as the mean ±S.E.M. For multiplecomparisons of different groups, the Kruskall-Wallis test for analysisof variance is used. The Dunn's test for comparison between twoindividual groups is then performed. A p value of less than 0.05 isaccepted as significant.

The embodiments disclosed herein are not to be limited in scope by thespecific embodiments disclosed in the examples which are intended asillustrations of a few aspects of the disclosed embodiments and anyembodiments that are functionally equivalent are encompassed by thepresent disclosure. Indeed, various modifications of the embodimentsdisclosed herein are in addition to those shown and described hereinwill become apparent to those skilled in the art and are intended tofall within the scope of the appended claims.

A number of references have been cited, the disclosures of which areincorporated herein by reference in their entirety.

1. A method for treating or preventing asthma, bronchitis, rhinitis,chronic obstructive pulmonary disease, lung inflammation or airwayhyperresponsiveness caused, induced or exacerbated by ozone, cold orexercise, comprising administering to a patient in need thereof aneffective amount of a compound having the formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R¹ issubstituted or unsubstituted C₁₋₆alkyl, substituted or unsubstitutedaryl, substituted or unsubstituted C₃₋₁₀cycloalkyl, substituted orunsubstituted C₃₋₁₀heterocycle or substituted or unsubstitutedC₃₋₁₀heteroaryl; R² is H, substituted or unsubstituted C₁₋₆alkyl,substituted or unsubstituted aryl, substituted or unsubstitutedC₃₋₁₀cycloalkyl, substituted or unsubstituted C₃₋₁₀heterocycle orsubstituted or unsubstituted C₃₋₁₀heteroaryl; and R³ is aryl substitutedwith one or more halogens or C₃₋₁₀heteroaryl substituted with one ormore halogens, wherein the aryl or C₃₋₁₀heteroaryl group is optionallyfurther substituted with one or more C₁₋₆alkyl, hydroxyl, hydroxyalkyl,alkoxy, alkoxyalkyl, amino, alkylamino, carboxy, aminocarbonyl, cyano,acylamino, alkanesulfonylamino, tetrazolyl, triazolyl or imidazolylgroups.
 2. The method of claim 1, wherein R¹ is substituted orunsubstituted aryl.
 3. The method of claim 1, wherein R¹ is substitutedor unsubstituted C₁₋₆alkyl.
 4. The method of claim 1, wherein R¹ issubstituted or unsubstituted C₃₋₁₀cycloalkyl.
 5. The method of claim 1,wherein R¹ is substituted or unsubstituted C₃₋₁₀heterocycle.
 6. Themethod of claim 1, wherein R¹ is substituted or unsubstitutedC₃₋₁₀heteroaryl.
 7. The method of claim 1, wherein R¹ is substitutedC₃₋₁₀cycloalkyl.
 8. The method of claim 7, wherein R¹ is C₃₋₁₀cycloalkylsubstituted with one or more C₁₋₆alkyl, hydroxy, hydroxyalkyl, alkoxy,alkoxyalkyl, amino, alkylamino, carboxy, aminocarbonyl, cyano,acylamino, alkanesulfonylamino, tetrazolyl, triazolyl or imidazolylgroups.
 9. The method of claim 1, wherein R² is substituted orunsubstituted aryl.
 10. The method of claim 1, wherein R² is substitutedor unsubstituted C₁₋₆alkyl.
 11. The method of claim 1, wherein R² issubstituted or unsubstituted C₃₋₁₀cycloalkyl.
 12. The method of claim11, wherein R² is cyclohexyl substituted with one or more C₁₋₆alkyl,hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy,aminocarbonyl, cyano, acylamino, alkanesulfonylamino, tetrazolyl,triazolyl or imidazolyl groups.
 13. The method of claim 11, wherein R²is cyclopentyl substituted with one or more C₁₋₆alkyl, hydroxy,hydroxyalkyl, alkoxy, alkoxyalkyl, amino, alkylamino, carboxy,aminocarbonyl, cyano, acylamino, alkanesulfonylamino, tetrazolyl,triazolyl or imidazolyl groups.
 14. The method of claim 1, wherein R² issubstituted or unsubstituted C₃₋₁₀heterocycle.
 15. The method of claim14, wherein R² is substituted or unsubstituted 3-oxetanyl,3-tetrahydrofuranyl, 4-tetrahydropyranyl, 4-piperidinyl,4-(1-acyl)-piperidinyl, 4-(1-alkanesulfonyl)piperidinyl, 3-pyrrolidinyl,3-(1-acyl)pyrrolidinyl, and 3-(1-alkanesulfonyl)pyrrolidinyl.
 16. Themethod of claim 14, wherein R² is substituted or unsubstituted sulfurcontaining C₃₋₁₀heterocycle.
 17. The method of claim 16, wherein thesulfur containing C₃₋₁₀heterocycle is 4-(1,1-dioxo)thiopyranyl or3-(1,1-dioxo)thiofuranyl.
 18. The method of claim 1, wherein R² issubstituted or unsubstituted C₃₋₁₀heteroaryl.
 19. The method of claim 1,wherein R³ is halogen substituted aryl.
 20. The method of claim 19,wherein R³ is fluoro substituted aryl.
 21. The method of claim 1,wherein R³ is halogen substituted C₃₋₁₀heteroaryl.
 22. The method ofclaim 21, wherein R³ is fluoro substituted C₃₋₁₀heteroaryl
 23. Themethod of claim 1, wherein R³ is

wherein: X is at each occurrence independently F, Cl, Br or I; R₆ isC₁₋₆alkyl, hydroxyl, hydroxyalkyl, alkoxy, alkoxyalkyl, amino,alkylamino, carboxy, aminocarbonyl, cyano, acylamino,alkanesulfonylamino, tetrazolyl, triazolyl or imidazolyl; m is aninteger ranging from 1 to 5; and p is an integer ranging from 1 to 4.24. The method of claim 23, wherein X is F, Cl or Br.
 25. The method ofclaim 23, wherein m is 1, 2 or 3 and X is F or Cl.
 26. The method ofclaim 23, wherein m is 2 or 3 and at least one X is F and at least one Xis Cl.